NF-κB inhibitors and uses thereof

ABSTRACT

A new class of imidazolines as 4-position esters with very potent anti-inflammatory as well as antimicrobial activity is described. The synthesis of these imidazolines includes a multicomponent reaction applicable to a combinatorial synthetic approach. The combination of these two key characteristics provides an effective therapeutic drug in the treatment of septic shock as well as many other inflammatory (arthritis and asthma) and infectious disorders. The use of this novel class of non-steroidal agents as anti-inflammatory agents (for the treatment of asthma, etc.), antibacterial agents, and antiseptic agents is described. The compounds are also useful in the treatment of tumors (such as cancers). The imidazolines are potent inhibitors of the transcription factor NF-κB as well as potent activity against the Gram (+) bacterium. The compositions are also useful for treating autoimmune diseases and for inhibiting rejection of organ and tissue transplants.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of co-pending U.S. patent applicationSer. No. 10/726,411, filed Dec. 3, 2003 now issued as U.S. Pat. No.7,528,161 on May 5, 2009, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/449,662, filed May 30, 2003 now issued as U.S.Pat. No. 7,345,078 on Mar. 18, 2008, which is a continuation-in-part ofU.S. patent application Ser. No. 10/347,323, filed Jan. 17, 2003 nowissued as U.S. Pat. No. 6,878,735 on Apr. 12, 2005, and which claimspriority to U.S. Provisional Patent Application Ser. No. 60/385,162,filed May 31, 2002.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

REFERENCE TO A “COMPUTER LISTING APPENDIX SUBMITTED ON A COMPACT DISC”

Not Applicable.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to novel multi-substituted 4-acid or esteror amide imidazolines and to a process for their preparation. Inparticular the present invention relates to the multi-substitutedimidazolines containing a 4-acid or an ester group which inhibit NF-κBor NF-κB kinase, are anti-inflammatory and/or antimicrobial and/orchemopotentiator and/or chemosensitizers of anticancer agents and/orimmune response inhibitors to foreign and endogenous NF-κB activators.The compositions are useful for treating inflammatory diseases,Alzheimer's disease, stroke atherosclerosis, restenosis, diabetes,glomerulophritis, cancer, Hodgkins disease, cachexia, inflammationassociated with infection and certain viral infections, includingacquired immune deficiency syndrome (AIDS), adult respiratory distresssyndrome, Ataxia Telangiestasia, and a variety of skin related diseases.The compositions are also useful for treating autoimmune diseases andfor inhibiting rejection of organ and tissue transplants.

(2) Description of Related Art

The mammalian nuclear transcription factor, NF-κB, is a multisubunitcomplex involved in the activation of gene transcription, including theregulation of apoptosis (programmed cell death) (Baeuerle, Henkel, Ann.Rev. Immunol., 12: 141-179 (1994); Baldwin, Ann. Rev. Immunol. 14,649-683 (1996)). NF-kB exists mainly as a homodimer (p50/p50) orheterodimer (p50/p65) in the cytoplasm in the form of an inactivecomplex with the inhibitory IkB protein. Many cellular stimuli includingantineoplastic agents (White, J. Biol. Chem. 272: 14914-14920 (1997);Baldwin, J. Clin. Invest. 107: 241-246 (2001); Hideshima et al., J.Biol. Chem. 277: 16639-16647 (2002); Bottero et al., Cancer Res. 61:7785-7791 (2001); Um et al., Oncogene 20: 6048-6056 (2001); Weldon etal., Surgery 130: 143-150 (2001); Arlt et al., Oncogene 20: 859-868(2001); Liu et al., J. Immunol. 166: 5407-5415 (2001); Kim et al.,Biochem. Biophys. Res. Commun. 273: 140-146 (2000)), viruses (HIV-1,HTLV-1), inflammatory cytokines (TNF-α, IL-1), phorbol esters, bacterialproducts (LPS), and oxidative stress, result in the IKK mediatedphosphorylation of IkB on serines 32 and 36, followed by ubiquitinationand subsequent degradation by the 26S proteosome (Baeuerle and Henkel,Ann. Rev. Immunol. 12: 141-179 (1994). Degradation of IκB ensures therelease of NF-kB. Upon release, NF-κB translocates into the nucleuswhere the subunits bind with specific DNA control elements and initiatesgene transcription. During translocation, additional proteinphosphorylation events are required for optimal gene transcription(Karin and Lin, Nat. Immunol. 3: 221-227 (2002); Zhong et al., Cell 89:413-424 (1997); Sizemore et al., Mol. Cell. Biol 19: 4798-4805 (1999);Madrid et al., Mol. Cell. Biol 20: 1626-1638 (2000)). Even though thekinases responsible for this phosphorylation event are not yet clearlyidentified, increasing evidence suggests the involvement of the cyclindependent kinase GSK-3 (Schwabe and Brenner, Am. J. Physiol.Gastrointest. Liver Physiol. 283: G204-211 (2002); Ali et al., Chem.Rev. 101: 2527-2540 (2001)). Inhibition of NF-κB mediated genetranscription can be accomplished through inhibition of phosphorylationof the inhibitory protein IκB, inhibition of IκB degradation, inhibitionof NF-κB (p50/p65) nuclear translocation, the inhibition of NF-κB-DNAbinding or NF-κB-mediated DNA transcription (for a comprehensive reviewon NF-κB inhibitors, see Epinat and Gilmore, Oncogene 18: 6896-6909(1999)). Genes regulated by NF-κB activation are a number of cytokines(TNF, IL-1, IL-2, IL-6, IL-8, iNOS), chemokines, cell adhesionmolecules, acute phase proteins, immunoregulatory proteins, eicosanoidmetabolizing enzymes, and anti-apoptotic genes.

NF-κB activation plays a role in cancer related disease. NF-κB isactivated by oncogenic ras (the most common defect in human tumors),TNF, ionizing radiation (radiation damage) and chemotherapeutic agents.Activation of NF-κB by these signals results in the upregulation ofanti-apoptotic cell signals and can therefore result in tumor cellresistance to chemotherapy. Inhibition of NF-κB is therefore a possibletreatment in sensitizing tumors to chemotherapeutic drugs and thepotential of novel cancer therapies. Related information on thistreatment is found in (Das and White, J. Biol. Chem. 272: 14914-14920(1997); Baldwin, J. Clin Invest. 107: 241-246 (2001); Hideshima et al.,J. Biol. Chem. 277: 16639-16647 (2002); Weldon et al., Surgery 130:143-150 (2001); Arlt et al., Oncogene 20: 859-868 (2001); Crinelli etal., Blood Cells Mol. Dis. 26: 211-222 (2000); Mayo and Baldwin,Biochim. Biophys. Acta 1470: M55-62 (2000); Adams, Curr. Opin. Chem.Biol. 6: 493-500 (2002); Boland, Biochem. Soc. Trans. 29: 674-678(2001); Chen et al., Am. J. Pathol. 159: 387-397 (2001); Cusack et al.,Drug Resist. Updat. 2: 271-273 (1999); Darnell, Jr., Nat. Rev. Cancer 2:740-749 (2002); Guttridge et al., Mol. Cell. Biol. 19: 5785-5799 (1999);Jones et al., Ann. Thorac. Surg. 70: 930-936 (2000); discussion 936-937;Orlowski et al., J. Clin. Oncol. 20: 4420-4427 (2002); Royds et al.,Mol. Pathol. 51: 55-61 (1998); Shah et al., J. Cell. Biochem. 82:110-122 (2001); Wang et al., Science, 274: 784-787 (1996)). NF-κBactivation also plays a significant role in inflammation disorders.NF-κB is activated by TNF and other pro-inflammatory cytokines.Inhibition of NF-κB activation by non-toxic inhibitors could thereforehave clinical use in the treatment of many inflammatory disorders,rheumatoid arthritis, inflammatory bowel disease, asthma, chronicobstructive pulmonary disease (COPD) osteoarthritis, osteoporosis andfibrotic diseases. Related information on this can be found in (Feldmannet al., Ann. Rheum. Dis. 61: Suppl 2, ii13-18 (2002); Gerard andRollins, Nat. Immunol. 2: 108-115 (2001); Hart et al., Am. J. Respir.Crit. Care Med. 158: 1585-1592 (1998); Lee and Burckart, J. Clin.Pharmacol. 38: 981-993 (1998); Makarov, Arthritis Res. 3: 200-206(2001); Manna et al., J. Immunol. 163: 6800-6809 (1999); Miagkov et al.,Proc. Natl. Acad. Sci. USA, 95, 13859-13864 (1998); Miossec, Cell Mol.Biol. (Noisy-1e-grand), 47: 675-678 (2001); Roshak et al., Curr. Opin.Pharmacol. 2: 316-321 (2002); Tak and Firestein, J. Clin. Invest. 107:7-11 (2001); Taylor, Mol. Biotechnol. 19: 153-168 (2001); Yamamoto andGaynor, J. Clin. Invest. 107: 135-142 (2001); Zhang and Ghosh, J.Endotoxin Res. 6: 453-457 (2000)).

NF-κB activation plays a significant role in immune disorders (Ghosh etal., Ann. Rev. Immunol. 16: 225-260 (1998)). Activation of the NF-κBresults in the active transcription of a great variety of genes encodingmany immunologically relevant proteins (Baeuerle and Henkel, Ann. Rev.Immunol. 12: 141-179 (1994); Daelemans et al., Antivir. Chem. Chemother.10: 1-14 (1999)). In the case of the human immunodeficiency virus (HIV)infection results in NF-κB activation, which results in regular viralpersistence (Rabson, A. B., Lin, H. C. Adv Pharmacol, 48, 161-207(2000); Pati, S., Foulke, J. S., Jr., Barabitskaya, O., Kim, J., Nair,B. C. et al. J Virol, 77, 5759-5773 (2003); Quivy, V., Adam, E.,Collette, Y., Demonte, D., Chariot, A. et al. J Virol, 76, 11091-11103(2002); Amini, S., Clavo, A., Nadraga, Y., Giordano, A., Khalili, K. etal. Oncogene, 21, 5797-5803 (2002); Takada, N., Sanda, T., Okamoto, H.,Yang, J. P., Asamitsu, K. et al. J Virol, 76, 8019-8030 (2002);Chen-Park, F. E.; Huang, D. B., Noro, B., Thanos, D., Ghosh, G. J BiolChem, 277, 24701-24708 (2002); Ballard, D. W. Immunol Res, 23, 157-166(2001); Baldwin, A. S., Jr. J Clin Invest, 107, 3-6 (2001); Calzado, M.A., MacHo, A., Lucena, C., Munoz, E. Clin Exp Immunol, 120, 317-323(2000); Roland, J., Berezov, A., Greene, M. I., Murali, R.,Piatier-Tonneau, D. et al. DNA Cell Biol, 18, 819-828 (1999); Boykins,R. A., Mahieux, R., Shankavaram, U. T., Gho, Y. S., Lee, S. F. et al. JImmunol, 163, 15-20 (1999); Asin, S., Taylor, J. A., Trushin, S., Bren,G., Paya, C. V. J Virol, 73, 3893-3903 (1999); Sato, T., Asamitsu, K.,Yang, J. P., Takahashi, N., Tetsuka, T. et al. AIDS Res HumRetroviruses, 14, 293-298 (1998)). HIV-1 replication is regulatedthrough an variety of viral proteins as well as cellular transcriptionfactors (in particular NF-κB) that interact with the viral long terminalrepeat (LTR) (Asin, S., Taylor, J. A., Trushin, S., Bren, G., Paya, C.V. J Virol, 73, 3893-3903 (1999)). HIV-1 is able to enter a latent statein which the integrated provirus remains transcriptionally silent. Theability to continue to infect cells latently aids the virus to establishpersistent infections and avoid the host immune system. The latent viruscan establish large reservoirs of genetic variants in T-cells residingin lymphoid tissue. In addition, a recent study implicates NF-κB withthe reactivation of latent HIV in T-cells in patents undergoingantiviral therapy (Finzi, D., Hermankova, M., Pierson, T., Carruth, L.M., Buck, C. et al. Science, 278, 1295-1300 (1997)). Relevant patent isthis area are EP 0931544 A2 to Baba et al. and WO 02/30423 A1 toCallahan et al.

Chronic airway inflammation as seen with asthma, is associated with theover expression of inflammatory proteins called cytokines. In addition,other inflammatory mediators, such as IL-1 and TNF, play a major role injoint diseases such as rheumatoid arthritis. All of these inflammatoryproteins are highly regulated by the nuclear transcription factor kappaB (NF-κB) (Yamamoto, Y., et al., J. Clin Invest 107 135-142 (2001); andHart, L. A., et al., Am J Respir Crit Care Med 158 1585-1592 (1998)).Inhibition of this regulatory protein or its kinase by anti-inflammatorydrugs has been shown to be effective in the treatment of these diseases(Yamamoto, Y., et al., J. Clin Invest 107 135-142 (2001); Coward, W. R.,et al., Clin Exp Allergy 28 Suppl 3, 42-46 (1998); Badger, A. M., etal., J. Pharmacol Exp Ther 290 587-593 (1999); Breton, J. J., et al., JPharmacol Exp Ther 282 459-466 (1997); Roshak, A., et al., J PharmacolExp Ther 283 955-961 (1997); Kopp, E., et al., Science 265 956-959(1994); Ichiyama, T., et al., Brain Res 911 56-61 (2001); Hehner, S. P.,et al., J Immunol 163 5617-5623 (1999); Natarajan, K., et al., Proc NatlAcad Sci USA 93 9090-9095 (1996); and Fung-Leung, W. P., et al.,Transplantation 60 362-368 (1995)). The common anti-inflammatory agent,aspirin, and aspirin-like drugs, the salicylates, are widely prescribedagents to treat inflammation and their effectiveness has been attributedto NF-κB inhibition. However, in order to treat chronic inflammations,the cellular levels of these salicylates need to be at very highconcentration and are generally prescribed at 1-3 miliMolar plasmaconcentrations (Science 265, 956-959 (1994)).

Since the discovery of penicillin, over 100 antibacterial agents havebeen developed to combat a wide variety of bacterial infections. Today,the clinically used antibacterial agents mainly consists of β-lactams(penicillins, carbapenems and cephalosporins), aminoglycosides,tetracyclines, sulfonamides, macrolides (erythromycin), quinolones, andthe drug of last resort: vancomycin (a glycopeptide). In recent years,many new strains of bacteria have developed resistance to these drugsthroughout the world. There is a need for new antimicrobials.

Invasive infection with Gram positive or Gram negative bacteria oftenresults in septic shock and death. Invasion of the blood stream by bothtypes of bacteria (Gram positive and Gram negative) causes sepsissyndrome in humans as a result of an endotoxin, Lipopolysaccharide (LPS)(H. Bohrer, J. Clin. Invest. 972-985 (1997)), that triggers a massiveinflammation response in the host. The mechanism by which LPS causedseptic shock is through the activation of the transcription factorNF-κB. Activation of this protein by its kinase initiates the massiverelease of cytokines resulting in a potentially fatal septic shock. Forexample, the pneumococcus bacteria is the leading cause of death with amortality rate of 40% in otherwise healthy elderly individuals andstaphylococcal infections are the major cause of bacteremia in UShospitals today. Septic shock, caused by an exaggerated host response tothese endotoxins often leads to multiple organ dysfunction, multipleorgan failure, and remains the leading cause of death in traumapatients. Inhibition of NF-kB activation by LPS would, therefore, betherapeutically useful in the treatment of Septic shock and otherbacterial infections.

There is considerable interest in modulating the efficacy of currentlyused antiproliferative agents to increase the rates and duration ofantitumor effects associated with conventional antineoplastic agents.Conventional antiproliferative agents used in the treatment of cancerare broadly grouped as chemical compounds which (1) affect the integrityof nucleic acid polymers by binding, alkylating, inducing strand breaks,intercalating between base pairs or affecting enzymes which maintain theintegrity and function of DNA and RNA; and (2) chemical agents that bindto proteins to inhibit enzymatic action (e.g. antimetabolites) or thefunction of structural proteins necessary for cellular integrity (e.g.,antitubulin agents). Other chemical compounds that have been identifiedto be useful in the treatment of some cancers include drugs which blocksteroid hormone action for the treatment of breast and prostate cancer,photochemically activated agents, radiation sensitizers and protectors.

Of special interest to this invention are those compounds that directlyaffect the integrity of the genetic structure of the cancer cells.Nucleic acid polymers such as DNA and RNA are prime targets foranticancer drugs. Alkylating agents such as nitrogen mustards,nitrosoureas, aziridine (such as mitomycin C) containing compoundsdirectly attack DNA. Metal coordination compounds such as cisplatin andcarboplatin similarly directly attack the nucleic acid structureresulting in lesions that are difficult for the cells to repair, which,in turn, can result in cell death. Other nucleic acid affectingcompounds include anthracycline molecules such as doxorubicin, whichintercalates between the nucleic acid base pairs of DNA polymers,bleomycin which causes nucleic acid strand breaks, fraudulentnucleosides such as pyrimidine and purine nucleoside analogs which areinappropriately incorporated into nucleic polymer structures andultimately cause premature DNA chain termination. Certain enzymes thataffect the integrity and functionality of the genome can also beinhibited in cancer cells by specific chemical agents and result incancer cell death. These include enzymes that affect ribonucleotidereductase (.e.g., hydroxyurea, gemcitabine), topoisomerase I (e.g.,camptothecin) and topoisomerase II (e.g. etoposide).

The topoisomerase enzymes affect the structure of supercoiled DNA,because most of the functions of DNA require untwisting. Topoisomerase I(top 1) untwists supercoiled DNA, breaking only one of the two strands,whereas topoisomerase II (top 2) breaks both.

Topoisomerase I inhibition has become important in cancer chemotherapythrough the finding that camptothecin (CPT), an alkaloid of plantorigin, is the best known inhibitor of top 1 and is a very potentanticancer agent. CPT is contained in a Chinese tree, Camptothecaacuminata. A number of analogs have become approved for commercial useto treat a number of tumor types. These include CPT-11 (irinotecan) andtopotecan.

While the clinical activity of camptothecins against a number of typesof cancers are demonstratable, improvements in tumor response rates,duration of response and ultimately patient survival are still sought.The invention described herein demonstrates the novel use which canpotentiate the antitumor effects of chemotherapeutic drugs, includingtopoisomerase I inhibitors, in particular, camptothecins.

Relevant Literature includes the following: Cancer ChemotherapeuticAgents, W. O. Foye, ed., (ACS, Washington, D.C.) (1995)); CancerChemotherapy Handbook, R. T. Dorr and D. D. VonHoff, (Appleton andLange, Norwalk, Conn.) (1994); and M. P. Boland, Biochemical SocietyTransactions (2001) volume 29, part 6, p 674-678. DNA damage signalingand NF-κB: implications for survival and death in mammalian cells.

NF-κB has been indicated to inhibit apoptosis (programmed cell death).Many clinically used chemotherapeutic agents (including the vincaalkaloids, vincristine and vinblastine, camptothecin and many others)have recently been shown to activate NF-κB resulting in a retardation oftheir cytotoxicity. This form of resistance is commonly referred to asNF-κB mediated chemoresistance. Inhibition of NF-κB has shown toincrease the sensitivity to chemotherapeutic agents of tumor cells andsolid tumors.

References: Cusack, J. C., Liu, F., Baldwin, A. S. Drug Resist Updat, 2,271-273 (1999); Mayo, M. W., Baldwin, A. S. Science, 274, 784-787(1996); Cusack, J. C., Jr., Liu, R., Baldwin, A. S., Jr. Cancer Res, 60,2323-2330 (2000). Brandes, L. M., Lin, Z. P., Patierno, S. R., Kennedy,K. A. Mol Pharmacol, 60, 559-567 (2001); Arlt, A., Vorndamm, J.,Breitenbroich, M., Folsch, U. R., Kalthoff, H. et al. Oncogene, 20,859-868 (2001). Cusack, J. C., Jr., Liu, R., Houston, M., Abendroth, K.,Elliott, P. J. et al. Cancer Res 61, 3535-3540 (2001).

The current invention describes the synthesis and application ofimidazolines as clinically important compounds. The imidazolines wereprepared via a new 1,2-dipolar cycloadditions reaction. 1,3 Dipolarcycloadditions reactions utilizing azlactones of “munchones” provide ageneral route for the synthesis of pyrroles and imidazoles (Hershenson,F. M. P., Synthesis 999-1001 (1988); Consonni, R. C., et al., J. chem.Research (S) 188-189 (1991); and Bilodeau, M. T. C., J. Org. Chem. 632800-2801 (1998)). This approach has not yet been reported for theimidazoline class of heterocycles. The synthetic and pharmacologicalinterest in efficient syntheses of imidazolines has fueled thedevelopment of several diverse synthetic approaches (Puntener, K., etal., J. Org Chem 65 8301-8306 (2000); Hsiao, Y. H., J. Org. Chem. 623586-3591 (1997)). Recently, Arndtsen et al reported synthesis ofsymmetrically substituted imidazoline-4-carboxylic acids via aPd-catalyzed coupling of an imine, acid chloride and carbon monoxide(Dghaym, R. D. D., et al., Angew. Chem. Int. Ed. Engl. 40 3228-3230(2001)). In addition, diastereoselective 1,3-dipolar cycloaddition ofazomethine ylides has been reported from amino acid esters withenantiopure sulfinimines to yield N-sulfinyl imidazolidines (Viso, A.,et al., J. Org. Chem. 62 2316-2317 (1997)).

U.S. Pat. No. 6,318,978 to Ritzeler et al describes 3,4-benzimidazoleswhich are structurally quite different than those of the presentinvention. They inhibit NF-κB kinase. As can be seen, activity isretained where there are numerous different substituents in theimidazoline and benzene rings. M. Karin, Nature immunology, 3, 221-227(2002); Baldwin, J. Clin. Invest., 3, 241-246 (2001); T. Huang et al, J.Biol. Chem., 275, 9501-9509 (2000); and J. Cusack and Baldwin, CancerResearch, 60, 2323-2330 (2000) describe the effect of activation ofNF-κB on cancer. U.S. Pat. Nos. 5,804,374 and 6,410,516 to Baltimoredescribe NF-κB inhibition which are incorporated by reference.

Patents of interest for the general methodology of inhibition are setforth in U.S. Pat. Nos. 5,821,072 to Schwartz et al and 6,001,563 toDeely et al.

SUMMARY OF THE INVENTION

The present invention relates to a method for inhibiting inflammation ina mammal which comprises administering a multi-substituted 4-acid or4-alkyl ester imidazoline to the mammal in an amount sufficient toinhibit the inflammation.

The present invention also relates to a method of inhibiting theactivation of the NF-κB protein by inhibition of the degradation of theinhibitory protein, I kappa B, or its kinases and the ability to inhibitNF-κB which comprises of contacting the protein or its activatingproteins with a multi-substituted 4-acid or 4-alkyl ester or amideimidazoline in an amount sufficient to inhibit activation of theprotein.

The present invention also relates to a method for inhibiting autoimmunediseases, certain viral infections, including acquired immune deficiencysyndrome (AIDS), adult respiratory distress syndrome, AtaxiaTelangiestasia and a variety of skin related diseases, includingpsoriasis, atopic dermatitis and ultraviolet radiation induced skindamage.

The present invention further relates to inhibiting an immune responseto a foreign NF-κB activator introduced into a mammal which makes thecompounds useful for treatment of autoimmune diseases and useful forinhibiting rejection of tissue, skin, and organ transplants.

The present invention further relates to a method of inhibiting a cancerwhich comprises contacting the cancer with a multi-substitutedimidazoline in an amount sufficient to inhibit the cancer.

The present invention relates to an imidazoline of the formula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to 14 ringmembers, and heterocyclic containing 5 to 12 ring members; X is selectedfrom the group consisting of O and S; and R₅ is selected from the groupconsisting of hydrogen, alkyl, acyl, aryl arylalkyl, heteroaryl, NH₂,NH—R₆ and

where R₆ and R₇ are selected from the group consisting of hydrogen,alkyl, aryl, arylalkyl, and heteroaryl and heterocyclic, which may bethe same or different.

Further the present invention relates to an imidazoline of the formula

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to 14 ringmembers, and heterocyclic containing 5 to 12 ring members; and whereinR₈ and R₉ and selected from the group consisting of hydrogen, alkyl,aryl, arylalkyl, heteroaryl and heterocyclic, which may be the same ordifferent.

Further, the present invention relates to a process for the preparationof an amino imidazoline which comprises reacting an imidazoline of theformula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to 14 ringmembers, and heterocyclic containing 5 to 12 ring members; X is selectedfrom the group consisting of O and S; and R₅ is selected from the groupconsisting of hydrogen, alkyl, acyl, aryl arylalkyl, heteroaryl, NH₂,NH—R₆ and

where R₆ and R₇ and selected from the group consisting of hydrogen,alkyl, aryl, arylalkyl, and heteroaryl and heterocyclic, which may bethe same or different, with an amine of the formula:

to produce a compound of the formula:

wherein R₈ and R₉ are selected from the group consisting of hydrogen,alkyl, acyl, arylalkyl and heteroalkyl, which may be the same ordifferent.

The present invention relates to an imidazoline of the formula

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to 14 ringmembers, and heterocyclic containing 5 to 12 ring members; and R₅ isselected from the group consisting of hydrogen and an alkyl group, allof which are optionally substituted.

The present invention particularly relates to an imidazoline of theformula:

wherein R₁, R₂, R₃ and R₄ are each individually selected from the groupconsisting of alkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to14 ring members, and heterocyclic containing 5 to 12 ring members; andR₅ is selected from the group consisting of hydrogen and an alkyl group,all of which are optionally substituted. Preferably R₁ is phenyl; R₄ isbenzyl; R₅ is lower alkyl containing 1 to 4 carbon atoms. Alsopreferably R₅ is ethyl; R₂ is lower alkyl containing 1 to 4 carbonatoms. Most preferably R₂ is methyl and R₃ is selected from the groupconsisting of phenyl and substituted phenyl.

The imidazoline (Compound 1) wherein R₁ is phenyl, R₂ is methyl, R₃ isphenyl, R₄ is benzyl and R₅ is H is a preferred compound. Theimidazoline (Compound 2) wherein R₁ is phenyl, R₂ is methyl, R₃ is4-methoxyphenyl, R₄ is benzyl and R₅ is H is a preferred compound. Theimidazoline (Compound 3) wherein R₁ is phenyl, R₂ is methyl, R₃ isphenyl, R₄ is 4-fluorophenyl and R₅ is H is a preferred compound. Theimidazoline (compound 4) wherein R₁ is phenyl, R₂ is phenyl, R₃ isphenyl, R₄ is benzyl and R₅ is H is a preferred compound. Theimidazoline (Compound 5) wherein R₁ is phenyl, R₂ is1H-indol-3-ylmethyl, R₃ is phenyl, R₄ is benzyl and R₅ is H is apreferred compound. The imidazoline (Compound 6) wherein R₁ is phenyl,R₂ is methyl, R₃ is pyridin-4-yl, R₄ is benzyl and R₅ is H is apreferred compound. The imidazoline (Compound 7) wherein R₁ is phenyl,R₂ is methyl, R₃ is phenyl, R₄ is H and R₅ is H is a preferred compound.The imidazoline (Compound 8) wherein R₁ is phenyl, R₂ is methyl, R₃ isethoxycarbonyl, R₄ is H and R₅ is H is a preferred compound. Theimidazoline (Compound 9) wherein R₁ is phenyl, R₂ is methyl, R₃ ispyridin-4-yl, R₄ is benzyl and R₅ is Et is a preferred compound. Theimidazoline (Compound 10) wherein R₁ is phenyl, R₂ is methyl, R₃ isphenyl, R₄ is benzyl and R₅ is Et is a preferred compound.

The present invention also relates to a process for the preparation ofimidazoline of the formula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to 14 ringmembers, and heterocyclic containing 5 to 12 ring members; and R₅ isselected from the group consisting of hydrogen and an alkyl group, allof which are optionally substituted, which comprises:

(a) reacting a reaction mixture of

-   -   (1) an oxazolone of the formula:

-   -   (2) a ketone of the formula:        R₃═O    -   ; and    -   (3) an amine of the formula:        H₂N—R₄        in the presence of trimethyl silyl chloride or an acid chloride        and a solvent for the reactants in the absence of water in the        presence of a non-reactive gas and at a temperature between        about 0 and 100° C. to produce the imidazoline; and

(b) separating the imidazoline from the reaction mixture. Theimidazoline can be esterified by reaction with an alcohol. Theimidazoline is most preferably esterified by reaction with the alcoholand sulfonyl dichloride.

The present invention relates to a method for inhibiting inflammation ina mammal which comprises administering an imidazoline of the formula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, aralkyl, heteroaryl containing 5 to 14 ring members,and heterocyclic containing 5 to 12 ring members; and R₅ is selectedfrom the group consisting of hydrogen and an alkyl group, all of whichare optionally substituted, to the mammal in an amount sufficient toinhibit the inflammation. Preferably the mammal is human. The mammal canbe a lower mammal. The administration can be oral, topical, or byinjection (such as intravenous) into the mammal.

The present invention also relates to a method for inhibiting amicroorganism which comprises:

administering an effective amount of a compound of the formula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, aralkyl, heteroaryl containing 5 to 14 ring members,and heterocyclic containing 5 to 12 ring members; and R₅ is selectedfrom the group consisting of hydrogen and an alkyl group, all of whichare optionally substituted, to inhibit the microorganism. The inhibitioncan be in vitro or in vivo. The administration can be to a lower mammalor to a human. The administration can be oral, by injection into themammal, or topical.

Further, the present invention relates to a method of inhibitingdegradation of a protein which is NF-κB or NF-κB kinase which comprisescontacting the protein with a compound of the formula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, aralkyl, heteroaryl containing 5 to 14 ring members,and heterocyclic containing 5 to 12 ring members; and R₅ is selectedfrom the group consisting of hydrogen and an alkyl group, all of whichare optionally substituted. The compounds are also useful in thetreatment of tumors (cancers) where NFkB is involved. The inhibition ispreferably in vivo.

The present invention further relates to a method for inhibiting animmune response to a foreign NF-κB activator introduced into a mammalwhich comprises administering an effective amount of an imidazoline ofthe formula:

wherein R₁, R₂, R₃ and R₄ are each individually selected from the groupconsisting of alkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to14 ring members, and heterocyclic containing 5 to 12 ring members; and,R₅ is selected from the group consisting of hydrogen and an alkyl group,all of which are optionally substituted, to the mammal so as to therebyinhibit the immune response to the foreign NF-κB activator.

The present invention further relates to a method for treating anautoimmune disease in a mammal without bringing on completeimmunodeficiency in the mammal which comprises administering aneffective amount of an imidazoline of the formula:

wherein R₁, R₂, R₃ and R₄ are each individually selected from the groupconsisting of alkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to14 ring members, and heterocyclic containing 5 to 12 ring members; and,R₅ is selected from the group consisting of hydrogen and an alkyl group,all of which are optionally substituted, to the mammal so as to treatthe autoimmune disease.

The present invention further relates to a method for inhibitingrejection of an organ transplanted into a mammal which comprisesadministering an effective amount of an imidazoline of the formula:

wherein R₁, R₂, R₃ and R₄ are each individually selected from the groupconsisting of alkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to14 ring members, and heterocyclic containing 5 to 12 ring members; and,R₅ is selected from the group consisting of hydrogen and an alkyl group,all of which are optionally substituted, to the mammal so as to inhibitrejection of the organ transplanted into the mammal.

The present invention further relates to a method for inhibitingreactivation of human immunodeficiency virus (HIV) in cells latentlyinfected with the HIV which comprises administering an effective amountof an imidazoline of the formula:

wherein R₁, R₂, R₃ and R₄ are each individually selected from the groupconsisting of alkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to14 ring members, and heterocyclic containing 5 to 12 ring members; and,R₅ is selected from the group consisting of hydrogen and an alkyl group,all of which are optionally substituted, to inhibit the reactivation ofthe HIV in the latently infected cells.

The present application relates to an imidazoline ester of the formula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to 14 ring memberswith O, N, S or combinations thereof, and heterocyclic containing 5 to12 ring members with O, N, S; and R₅ a group which provides the ester ofthe imidazoline.

The present invention further relates to an imidazoline ester of theformula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to 14 ring memberswith O, N, S or combinations thereof, and heterocyclic containing 5 to12 ring members with O, N or S or combinations thereof; and R₅ a groupwhich provides the ester of the imidazoline; and R₅ is an ester groupcontaining 1 to 15 carbon atoms which are alkyl, cycloalkyl, aryl,heteroaryl comprising O, N or S or combinations thereof and heterocycliccomprising O, N, S or combinations thereof and wherein the carbon atomsare optionally substituted with a halogen.

The present invention also relates to an imidazoline ester of theformula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to 14 ring memberswith O, N, S or combinations thereof, and heterocyclic containing 5 to12 ring members with O, N or S or combinations thereof; and wherein inthe ester group, with R₁₁ and R₁₂ are selected from the group consistingof a hydrogen, alkyl, aryl, arylalkyl and a halogen, and R₆ to R₁₀ areselected from the group consisting of hydrogen, halogen, alkyl halide,ether, cyclic ether, cyclic alkyl, aryl or acyl, amine, hydroxyl andheterocyclic or heteroaryl rings with O, N or S or combinations thereofcomprising 5 to 14 carbon atoms.

The present invention relates to an imidazoline of the formula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to 14 ring memberswith O, N, S or combinations thereof, and heterocyclic containing 5 to12 ring members with O, N or S or combinations thereof; and R₅ is agroup which provides the ester of the imidazoline, wherein R₅ and R₆ areselected from the group consisting of hydrogen, alkyl, aryl, arylalkyland halogen and wherein R₇ is selected from the group consisting of aryland heterocyclic group containing one or more N, S, or O or combinationsthereof comprising 5 to 14 carbon atoms.

The present invention also relates to a method for inhibitinginflammation in a mammal which comprises administering an imidazolineester of the formula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofaryl, arylalkyl, heteroaryl containing 5 to 14 ring members, andheterocyclic containing 5 to 12 ring members; and R₅ is a group whichprovides the ester, all of which are optionally substituted, to themammal in an amount sufficient to inhibit the inflammation.

The present invention also relates to a method for inhibiting amicroorganism which comprises:

administering an effective amount of an imidazoline ester of theformula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to 14 ringmembers, and heterocyclic containing 5 to 12 ring members; and R₅ is agroup which provides the ester, all of which are optionally substituted,to inhibit the microorganism.

The present invention relates to a method of inhibiting degradation of aprotein which is NF-κB or NF-κB kinase which comprises contacting theprotein with a imidazoline ester of the formula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to 14 ringmembers, and heterocyclic containing 5 to 12 ring members; and R₅ is agroup which provides the ester, all of which are optionally substituted.

The present invention also relates to a method for inhibitinginflammation in a mammal which comprises administering amulti-substituted 4-acid or 4-alkyl ester imidazoline to the mammal inan amount sufficient to inhibit the inflammation.

The present invention also relates to a method of inhibiting degradationof a protein which is NF-κB or NF-κB kinase which comprises contactingthe protein with a multi-substituted imidazoline ester in an amountsufficient to inhibit degradation of the protein.

The present invention also relates to a method of inhibiting a cancerwhich comprises contacting the cancer with a multi-substitutedimidazoline ester in an amount sufficient to inhibit the cancer.

The present invention also relates to a method for inhibiting a tumor orcancer in a mammal which comprises administering an imidazoline ester ofthe formula:

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofaryl, arylalkyl, heteroaryl containing 5 to 14 ring members, andheterocyclic containing 5 to 12 ring members; and R₅ is a group whichprovides the ester, all of which are optionally substituted, to themammal in an amount sufficient to inhibit the tumor or cancer.

The present invention relates to a composition which comprises animidazoline of the formula

wherein R₁, R₂, R₃ and R₄ are selected from the group consisting ofalkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to 14 ringmembers, and heterocyclic containing 5 to 12 ring members; and R₅ is agroup which provides the ester, all of which are optionally substituted;and

(b) a drug which inhibits growth of the tumor or cancer.

The present invention relates to a method for inhibiting an immuneresponse to a foreign NF-κB activator introduced into a mammal whichcomprises:

administering an effective amount of an imidazoline ester of theformula:

wherein R₁, R₂, R₃ and R₄ are each individually selected from the groupconsisting of alkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to14 ring members, and heterocyclic containing 5 to 12 ring members; and,R₅ is a group which provides the ester, all of which are optionallysubstituted, to the mammal so as to thereby inhibit the immune responseto the foreign NF-κB activator.

The present invention also relates to a method for treating anautoimmune disease in a mammal without bringing on completeimmunodeficiency in the mammal which comprises:

administering an effective amount of an imidazoline ester of theformula:

wherein R₁, R₂, R₃ and R₄ are each individually selected from the groupconsisting of alkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to14 ring members, and heterocyclic containing 5 to 12 ring members; and,R₅ is a group which provides the ester, all of which are optionallysubstituted, to the mammal so as to treat the autoimmune disease.

The present invention further relates to a method for inhibitingrejection of an organ transplanted into a mammal which comprises:

administering an effective amount of an imidazoline ester of theformula:

wherein R₁, R₂, R₃ and R₄ are each individually selected from the groupconsisting of alkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to14 ring members, and heterocyclic containing 5 to 12 ring members; and,R₅ is a group which provides the ester, all of which are optionallysubstituted, to the mammal so as to inhibit rejection of the organtransplanted into the mammal.

The present invention also relates to a method for inhibitingreactivation of human immunodeficiency virus (HIV) in cells latentlyinfected with the HIV which comprises:

administering an effective amount of an imidazoline ester of theformula:

wherein R₁, R₂, R₃ and R₄ are each individually selected from the groupconsisting of alkyl, acyl, aryl, arylalkyl, heteroaryl containing 5 to14 ring members, and heterocyclic containing 5 to 12 ring members; and,R₅ is a group which provides the ester, all of which are optionallysubstituted, to inhibit the reactivation of the HIV in the latentlyinfected cells.

In further embodiments of the above methods, R₁ is phenyl, R₄ is benzyl,R₅ is lower alkyl containing 1 to 4 carbon atoms, R₅ is ethyl, R₂ islower alkyl containing 1 to 4 carbon atoms, R₂ is methyl and R₃ isselected from the group consisting of phenyl and substituted phenyl, orcombinations of the above.

R₁ is

(1) phenyl, mono- or disubstituted independently of one another by

(1) (1)—CN;

(1)(2)—NO₂;

(1) (3)—O—(C₁-C₄)-alkyl;

(1) (4)—NH₂; or

(1) (5)—(C₁-C₄)-alkyl-NH₂;

(1)(6)—x, wherein x is a halogen.

(2) heteroaryl having 5 to 14 ring members, in which the heteroaryl isunsubstituted or mono-, di-, or trisubstituted independently of oneanother by —N—R¹⁴, in which R¹⁴ is —(C₁-C₆)-alkyl, —(C₃-C₆)-cycloalkyl,phenyl, halogen, —OH, or —(C₁-C₄)-alkyl; or

(3) a heterocycle having 5 to 12 ring members, in which the heterocycleis unsubstituted or mono-, di-, or trisubstituted independently of oneanother by —N—R¹⁴, in which R¹⁴ is —(C₁-C₆)-alkyl, —(C₃-C₆)-cycloalkyl,phenyl, halogen, —OH, or —(C₁-C₄)-alkyl.

The term “halogen” is understood as meaning fluorine, chlorine, bromine,or iodine. The term “aryl” is understood as meaning aromatic hydrocarbongroups having 6 to 14 carbon atoms in the ring. (C₆-C₁₄)-Aryl groupsare, for example, phenyl, naphthyl, for example, 1-naphthyl, 2-naphthyl,biphenylyl, for example, 2-biphenylyl, 3-biphenylyl, and 4-biphenylyl,anthryl, or fluorenyl. Biphenylyl groups, naphthyl groups, and, inparticular, phenyl groups are preferred aryl groups. Aryl groups, inparticular phenyl groups, can be mono-substituted or polysubstituted,preferably monosubstituted, disubstituted, or trisubstituted, byidentical or different groups, preferably by groups selected from(C₁-C₈)-alkyl, in particular (C₁-C₄)-alkyl, (C₁-C₈)-alkoxy, inparticular (C₁-C₄)-alkoxy, halogen, nitro, amino, trifluoromethyl,hydroxyl, hydroxy-(C₁-C₄)-alkyl such as hydroxymethyl, 1-hydroxyethyl,or 2-hydroxyethyl, methylenedioxy, ethylenedioxy, formyl, acetyl, cyano,hydroxycarbonyl, aminocarbonyl, (C₁-C₄)-alkoxycarbonyl, phenyl, phenoxy,benzyl, benzyloxy, or tetrazolyl. Further, when aryl is phenyl, phenylis optionally mono- or disubstituted independently of one another by—CN, —NO₂, —O—(C₁-C₄)-alkyl, —N(R¹¹)₂, —NH—C(O)—R¹¹, —S(O)_(x)R¹¹, inwhich x is the integer 0, 1, or 2, —C(O)—R¹¹, in which R¹¹ is as definedabove, or —(C₁-C₄)-alkyl-NH₂. The same applies, for example, to groupssuch as arylalkyl or arylcarbonyl. Arylalkyl groups are, in particular,benzyl and also 1- and 2-naphthylmethyl, 2-, 3-, and 4-biphenylylmethyl,and 9-fluorenylmethyl. Substituted arylalkyl groups are, for example,benzyl groups and naphthylmethyl groups substituted in the aryl moietyby one or more (C₁-C₈)-alkyl groups, in particular (C₁-C₄)-alkyl groups,for example, 2-, 3-, and 4-methylbenzyl, 4-isobutylbenzyl,4-tert-butylbenzyl, 4-octylbenzyl, 3,5-dimethylbenzyl,pentamethylbenzyl, 2-, 3-, 4-, 5-, 6-, 7-, and8-methyl-1-naphthylmethyl, 1-, 3-, 4-, 5-, 6-, 7-, and8-methyl-2-naphthylmethyl, by one or more (C₁-C₈)-alkoxy groups, inparticular (C₁-C₄)-alkoxy groups, benzyl groups, and naphthylmethylgroups substituted in the aryl moiety for example, 4-methoxybenzyl,4-neopentyloxybenzyl, 3,5-dimethoxybenzyl, 3,4-methylenedioxybenzyl,2,3,4-trimethoxybenzyl, nitrobenzyl groups, for example, 2-, 3-, and4-nitrobenzyl, halobenzyl groups, for example, 2-, 3-, and 4-chloro- and2-, 3-, and 4-fluorobenzyl, 3,4-dichlorobenzyl, pentafluorobenzyl,trifluoromethylbenzyl groups, for example, 3- and4-trifluoromethylbenzyl, or 3,5-bis(trifluoromethyl)benzyl.

In monosubstituted phenyl groups, the substituent can be located in the2-position, the 3-position, or the 4-position. Disubstituted phenyl canbe substituted in the 2,3-position, the 2,4-position, the 2,5-position,the 2,6-position, the 3,4-position, or the 3,5-position. Intrisubstituted phenyl groups, the substituents can be located in the2,3,4-position, the 2,3,5-position, the 2,4,5-position, the2,4,6-position, the 2,3,6-position, or the 3,4,5-position.

The explanations for the aryl groups apply accordingly to divalentarylene groups, for example, to phenylene groups that can be present,for example, as 1,4-phenylene or as 1,3-phenylene.

Phenylene-(C₁-C₆)-alkyl is in particular phenylenemethyl (—C₆H₄—CH₂—)and phenyleneethyl. (C₁-C₆). Alkylenephenyl is in particularmethylenephenyl (—CH₂—C₆H₄—). Phenylene-(C₁-C₆)-alkenyl is in particularphenyleneethenyl and phenylenepropenyl.

The expression “heteroaryl having 5 to 14 ring members” represents agroup of a monocyclic or polycyclic aromatic system having 5 to 14 ringmembers, which contains 1, 2, 3, 4, or 5 heteroatoms as ring members.Examples of heteroatoms are N, O, and S. If a number of heteroatoms arecontained, these can be identical or different. Heteroaryl groups canlikewise be monosubstituted or polysubstituted, preferablymonosubstituted, disubstituted, or trisubstituted, by identical ordifferent groups selected from (C₁-C₈)-alkyl, in particular(C₁-C₄)-alkyl, (C₁-C₈)-alkoxy, in particular (C₁-C₄)-alkoxy, halogen,nitro, —N(R¹¹)₂, trifluoromethyl, hydroxyl, hydroxy-(C₁-C₄)-alkyl suchas hydroxymethyl, 1-hydroxyethyl, or 2-hydroxyethyl, methylenedioxy,formyl, acetyl, cyano, hydroxycarbonyl, aminocarbonyl,(C₁-C₄)-alkoxycarbonyl, phenyl, phenoxy, benzyl, benzyloxy, ortetrazolyl. Heteroaryl having 5 to 14 ring members preferably representsa monocyclic or bicyclic aromatic group which contains 1, 2, 3, or 4, inparticular 1, 2, or 3, identical or different heteroatoms selected fromN, O, and S, and which can be substituted by 1, 2, 3, or 4, inparticular 1, 2, or 3, identical or different substituents selected from(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, fluorine, chlorine, nitro, —N(R¹¹)₂,trifluoromethyl, hydroxyl, hydroxy (C₁-C₄)-alkyl,(C₁-C₄)-alkoxycarbonyl, phenyl, phenoxy, benzyloxy, and benzyl.Heteroaryl particularly preferably represents a monocyclic or bicyclicaromatic group having 5 to 10 ring members, in particular a 5-memberedor 6-membered monocyclic aromatic group which contains 1, 2, or 3, inparticular 1 or 2, identical or different heteroatoms selected from N,O, and S, and can be substituted by 1 or 2 identical or differentsubstituents selected from (C₁-C₄)-alkyl, halogen, hydroxyl, —N(R¹¹)₂,(C₁-C₄)-alkoxy, phenyl, phenoxy, benzyloxy, and benzyl. R¹¹ is asdefined in substituent R⁹ of formula I.

The expression “heterocycle having 5 to 12 ring members” represents amonocyclic or bicyclic 5-membered to 12-membered heterocyclic ring thatis partly saturated or completely saturated. Examples of heteroatoms areN, O, and S. The heterocycle is unsubstituted or substituted on one ormore carbons or on one or more heteroatoms by identical or differentsubstituents. These substituents have been defined above for the radicalheteroaryl. In particular, the heterocyclic ring is monosubstituted orpolysubstituted, for example, monosubstituted, disubstituted,trisubstituted, or tetrasubstituted, on carbons by identical ordifferent groups selected from (C₁-C₈)-alkyl, for example,(C₁-C₄)-alkyl, (C₁-C₈)-alkoxy, for example, (C₁-C₄)-alkoxy such asmethoxy, phenyl-(C₁-C₄)-alkoxy, for example, benzyloxy, hydroxyl, oxo,halogen, nitro, amino, or trifluoromethyl, and/or it is substituted onthe ring nitrogens in the heterocyclic ring by (C₁-C₈)-alkyl, forexample, (C₁-C₄)-alkyl such as methyl or ethyl, by optionallysubstituted phenyl or phenyl-(C₁-C₄)-alkyl, for example, benzyl.Nitrogen heterocycles can also be present as N-oxides or as quaternarysalts.

Examples of the expressions heteroaryl having 5 to 14 ring members orheterocycle having 5 to 12 ring members are groups which are derivedfrom pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole,thiazole, isothiazole, tetrazole, 1,3,4-oxadiazole,1,2,3,5-oxathiadiazole-2-oxides, triazolones, oxadiazolones,isoxazolones, oxadiazolidinediones, triazoles which are substituted byF, CN, CF₃, or COO—(C₁-C₄)-alkyl, 3-hydroxypyrrole-2,4-diones,5-oxo-1,2,4-thiadiazoles, pyridine, pyrazine, pyrimidine, indole,isoindole, indazole, phthalazine, quinoline, isoquinoline, quinoxaline,quinazoline, cinnoline, carboline, and benzo-fused, cyclopenta-,cyclohexa-, or cyclohepta-fused derivatives of these heterocycles.Particularly preferred groups are 2- or 3-pyrrolyl, phenylpyrrolyl suchas 4- or 5-phenyl-2-pyrrolyl, 2-furyl, 2-thienyl, 4-imidazolyl,methylimidazolyl, for example, 1-methyl-2,4-, or 5-imidazolyl,1,3-thiazol-2-yl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-, 3-, or4-pyridyl-N-oxide, 2-pyrazinyl, 2-, 4-, or 5-pyrimidinyl, 2-, 3-, or5-indolyl, substituted 2-indolyl, for example, 1-methyl-, 5-methyl-,5-methoxy-5-benzyloxy-, 5-chloro-, or 4,5-dimethyl-2-indolyl,1-benzyl-2- or -3-indolyl, 4,5,6,7-tetrahydro-2-indolyl,cyclohepta[b]-5-pyrrolyl, 2-, 3-, or 4-quinolyl, 1-, 3-, or4-isoquinolyl, 1-oxo-1,2-dihydro-3-isoquinolyl, 2-quinoxalinyl,2-benzofuranyl, 2-benzothienyl, 2-benzoxazolyl, or benzothiazolyl, ordihydropyridinyl, pyrrolidinyl, for example, 2- or3-(N-methylpyrrolidinyl), piperazinyl, morpholinyl, thiomorpholinyl,tetrahydrothienyl, or benzodioxolanyl.

Thus methods and compositions are provided for the treatment of a hostwith a cellular proliferative disease, particularly a neoplasia. In thesubject methods, pharmaceutically acceptable imidazolines and anantiproliferative agent are administered, preferably systemically.

Methods and compositions are provided for the treatment of a host with acellular proliferative disease, particularly a neoplasia. In the subjectmethods, a pharmaceutically acceptable imidazoline is administered,preferably systemically, in conjunction with an antiproliferative agentto improve the anticancer effects. In a preferred embodiment, theimidazoline provides a chemopotentiator effect.

A chemical agent is a chemopotentiator when it enhances the effect of aknown antiproliferative drug in a more than additive fashion relative tothe activity of the chemopotentiator or antiproliferative agent usedalone. In some cases, a chemosensitizing effect may be observed. This isdefined as the effect of use of an agent that if used alone would notdemonstrate significant antitumor effects but would improve theantitumor effects of an antiproliferative agent in a more than additivefashion than the use of the antiproliferative agent by itself.

As used herein, the term imidazoline includes all members of thatchemical family including the forms and analogs thereof. The imidazolinefamily is defined by chemical structure as the ring structurespreviously described.

As used herein, antiproliferative agents are compounds, which inducecytostasis or cytotoxicity. Cytostasis is the inhibition of cells fromgrowing while cytotoxicity is defined as the killing of cells. Specificexamples of antiproliferative agents include: antimetabolites, such asmethotrexate, 5-fluorouracil, gemcitabine, cytarabine; anti-tubulinprotein agents such as the vinca alkaloids, paclitaxel, colchicine;hormone antagonists, such as tamoxifen, LHRH analogs; and nucleic aciddamaging agents such as the alkylating agents melphalan, BCNU, CCNU,thiotepa, intercalating agents such as doxorubicin and metalcoordination complexes such as cisplatin and carboplatin. Preferably thedrug is a topoisomerase II inhibitor such as daunomycin.

Thus methods and compositions are provided for the treatment of a hostwith a cellular proliferative disease, particularly a neoplasia. In thesubject methods, pharmaceutically acceptable imidazolines and anantiproliferative agent are administered, preferably systemically.

Methods and compositions are further provided for the treatment of ahost with an autoimmune disease or an organ transplant or skin graft. Inthe subject methods, a pharmaceutically acceptable imidazoline isadministered, preferably systemically, optionally in conjunction withone or more anti-autoimmune or anti-rejection agents to improve theinhibition of the immune response involved in the autoimmune disease ortransplant or graft.

OBJECTS

It is an object of the present invention to provide novel compoundswhich inhibit immune responses to foreign NF-κB activators introducedinto a mammal.

It is also an object of the present invention to provide novel compoundswhich inhibit immune responses involved in rejection of organstransplanted into a mammal.

It is further an object of the present invention to provide novelcompounds which inhibit the immune response involved in autoimmunediseases in a mammal which involve NF-κB activation.

It is a further still object of the present invention to inhibit HIV byinhibiting NF-κB translocation to the cell nucleus of cells infectedwith HIV.

It is a further still object of the present invention to inhibit immuneresponses to foreign NFκB activators such as those involved in organtransplants or immune responses which are involved in autoimmunediseases without bringing on complete immunodeficiency in the mammal.

It is a further still object of the present invention to provide novelcompounds which are anti-inflammatory, antimicrobial and inhibit NFκB orNFκB kinase.

It is a further still object of the present invention to provide forinhibition of cancers by inhibition of chemoresistance.

It is a further still object of the present invention to provide a novelprocess for the preparation of such compounds.

These and other objects of the present invention will becomeincreasingly apparent with reference to the following drawings andpreferred embodiments.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of compounds 1 to 20.

FIG. 2 shows the x-ray crystal structure of compound 1 which isrepresentative.

FIG. 3 is a EMSA of nuclear extracts with imidazolines 8-10. Lane 1, DNAonly (control); lane 2, DNA, nuclear extract (10 μg) with p50 homodimer(control); lane 3, DNA, nuclear extract (10 μg) with PMA activation(control); lane 4, DNA, nuclear extract (10 μg) with no PMA activation(control); lane 5, DNA, nuclear extract (10 μg) after PMA activationwith compound 8 (1.0 μM); lane 6, DNA, nuclear extract (10 μg) after PMAactivation with compound 8 (0.1 μM); lane 7, DNA, nuclear extract (10μg) after PMA activation with compound 9 (1.0 μM); lane 8, DNA, nuclearextract (10 μg) after PMA activation with compound 9 (0.1 μM); lane 9,DNA, nuclear extract (10 μg) after PMA activation with compound 10 (0.1μM); lane 10, DNA, nuclear extract (10 μg) after PMA activation withcompound 10 (0.1 μM).

FIGS. 4A and 4B show tumor growth delay with compounds 4 and 6.

FIG. 5 shows the synthesis of imidazoline scaffolds 28-33.

FIG. 6 shows the structure of novel imidazolines 28-50. 37 and 38 areafter esterification with (COCl)₂, EtOH. 39 is after hydrogenation withH₂, 5% Pd/C.

FIG. 7 shows the inhibition of nuclear translocation of NF-κB bycompound 32 as measured by a p65 ELISA.

FIG. 8 shows the inhibition of nuclear translocation of NF-κB bycompounds 28-33 as measured by a p65 ELISA.

FIG. 9 shows that cell death over time in response to compound 32 isinsignificant.

FIG. 10A shows compounds 28, 32, and 33 are not toxic to mice.

FIG. 10B shows compounds 29 and 31 are not toxic to mice.

FIG. 11 shows an EMSA assay for NF-κB activation by camptothecin. Lane1: NF-κB consensus oligonucleotide (0.16 pmol/λ); Lane 2: NF-κBconsensus oligo (0.16 pmol/λ)+Nuclear extract (PMA/PHA, 20 μg); Lane 3:NF-κB consensus oligo (0.16 pmol/λ)+Nuclear extract (PMA/PHA, 20μg)+Antibody p65; Lane 4: NF-κB consensus oligo (0.16 pmol/λ)+Nuclearextract (−PMA/−PHA, 20 μg); Lane 5: NF-κB consensus oligo (0.16pmol/λ)+Nuclear extract (10 μM CPT, 20 μg); Lane 6: NF-κB consensusoligo (0.16 pmol/λ)+Nuclear extract (1 μM CPT, 20 μg); Lane 7: NF-κBconsensus oligo (0.16 pmol/λ)+Nuclear extract (0.1 μM CPT, 20 μg); Lane8: NF-κB consensus oligo (0.16 pmol/λ)+Nuclear extract (0.01 μM CPT, 20μg). All incubations with CPT were performed for 2 hours. The positivecontrol with PMA/PHA was incubated for 4 hours.

FIG. 12 shows an EMSA assay for inhibition of CPT activated NF-κB byimidazoline 32. Lane 1: NF-κB consensus oligonucleotide (0.16 pmol/λ);Lane 2: NF-κB consensus oligo (0.16 pmol/λ)+nuclear extract (PMA/PHA);Lane 3: NF-κB consensus oligo (0.16 pmol/λ)+nuclear extract(PMA/PHA)+Antibody p65; Lane 4: NF-κB consensus oligo (0.16pmol/λ)+nuclear extract (−PMA/−PHA); Lane 5: NF-κB consensus oligo (0.16pmol/λ)+nuclear extract (0.1 μM CPT); Lane 6: NF-κB consensus oligo(0.16 pmol/λ)+nuclear extract (0.1 μM CPT+5 μM PDTC); Lane 7: NF-κBconsensus oligo (0.16 pmol/λ)+nuclear extract (0.1 μM CPT+10 μM 32);Lane 8: NF-κB consensus oligo (0.16 pmol/λ)+nuclear extract (0.1 μMCPT+1 μM 32); Lane 9: NF-κB consensus oligo (0.16 pmol/λ)+nuclearextract (0.1 μM CPT+0.1 μM 32); Lane 10: NF-κB consensus oligo (0.16pmol/λ)+nuclear extract (0.1 μM CPT+0.01 μM 32). All incubations withCPT were performed for 2 hours. The positive control with PMA/PHA wasincubated for 4 hours.

FIG. 13A shows the sensitization of CEM cells towards camptothecin by 1μM imidazoline 31.

FIG. 13B shows the sensitization of CEM cells towards camptothecin by0.1 μM imidazoline 31.

FIG. 13C shows the sensitization of CEM cells towards camptothecin by0.01 μM imadizoline 31.

FIG. 13D shows the sensitization of CEM cells towards camptothecin by 1μM imidazoline 30.

FIG. 13E shows the sensitization of CEM cells towards camptothecin by0.1 μM imidazoline 30.

FIG. 13F shows the sensitization of CEM cells towards camptothecin by0.01 μM imidazoline 30.

FIG. 14 is a chart showing the dose dependent enhancement of apoptosismeasured after 48 hours in combinatorial treatment of CPT (0.1 μM) withvarying concentrations of imidazoline 32.

FIG. 15A shows the chemopotentiation of cis-platin by imidazoline 32. ●is 0.1 μM cis-platin and 0.1 μM imidiazoline 32; ▴ is 1.0 μM cis-platin;▪ is 0.01 μM cis-platin; ♦ is 10 μM imidazoline 32.

FIG. 15B is a chart showing dose responses of imidazoline 32 with 0.1 μMcis-platin.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

The present invention provides a method for inhibiting pathologicalactivation of the transcription factor NF-kappaB (NF-κB) byimidazolines. These agents were synthesized and found to be potentnon-toxic inhibitors of NF-κB. Such compounds may be used in thetreatment of diseases, in which activation of the NF-κB signalingpathway is involved. Inhibition of NF-κB activation inhibits thetranscription of genes related to a variety of inflammatory diseasessuch as: rheumatoid arthritis, inflammatory bowel disease, asthma,chronic obstructive pulmonary disease (COPD) osteoarthritis,osteoporosis and fibrotic diseases. Inhibition of NF-κB is useful in thetreatment of autoimmune diseases including systemic lupus eythematosus,multiple sclerosis, psoriatic arthritis, alkylosing spondylitis, andtissue and organ rejection. In addition, inhibition of NF-κB is usefulin the treatment of Alzheimer's disease, stroke atherosclerosis,restenosis, diabetes, glomerulophritis, cancer, Hodgkins disease,cachexia, inflammation associated with infection and certain viralinfections, including acquired immune deficiency syndrome (AIDS), adultrespiratory distress syndrome, Ataxia Telangiestasia and a variety ofskin related diseases, including psoriasis, atopic dermatitis andultraviolet radiation induced skin damage. In particular embodiments,the imidazolines herein have the ability to inhibit an immune responseto a foreign NF-κB activator introduced into a mammal which makes thecompounds useful for treatment of autoimmune diseases and useful forinhibiting rejection of tissue, skin, and organ transplants.

Preferred compounds are shown in FIGS. 1 and 6. The stereopositioning isshown in FIG. 2. The combination of these two key characteristics makesthis class of imidazolines an extremely effective therapeutic drug totreat inflammatory diseases, cancers, autoimmune diseases and to inhibitrejection of transplanted organs, tissues, and grafted skin. Theobjective of this invention is the use of multi-substituted imidazolinesfor therapeutic use as (1) anti-inflammatory agents (for example in thetreatment of asthma and rheumatoid arthritis), (2) antibacterial agents,including antiseptic agents, (3) anticancer agents and potentiators ofanticancer drugs such as cisplatin and the like, (4) anti-autoimmuneagents (for example, for the treatment of autoimmune diseases such assystemic lupus eythematous, multiple sclerosis, psoriatic arthritis,alkylosing spondylitis) and (5) anti-rejection agents for use in organtransplant and skin graft procedures with or without otheranti-rejection compounds to inhibit rejection of the transplanted organor grafted skin.

The compounds of the present invention are very potent inhibitors ofNF-κB in vitro (less than 0.1 μM concentrations) and preliminaryexperiments in cells have indicated that the compounds are not cytotoxicover a 72 hour time period. Several of the imidazolines indicatedantimicrobial activity against several strains of bacteria with MIC's of50 μg/mL.

The present invention also relates to the synthesis of the first classof imidazoline-type NF-κB inhibitors. The imidazolines were prepared viaa novel highly diastereoselective multicomponent synthesis using aminoacid derived oxazolidinones as general templates.

The general procedure for synthesis of imidazoline-4-carboxylic acids isas follows. A solution of aldehyde (for example 0.57 mmol), amine (forexample 0.57 mmol) in dry CH₂Cl₂ (10 mL) was refluxed under N₂ for 2hours. A solution of the oxazolone (for example 0.57 mmol) in dry CH₂Cl₂(for example 5 mL) was added and the mixture was refluxed under N₂ for 6hours, and then stirred overnight at room temperature. The product waspreferably either precipitated out from 1:1 CH₂Cl₂ or isolated aftersilica gel chromatography with 4:1 EtOAc/MeOH.

This is a novel highly diastereoselective multicomponent one-potsynthesis of aryl, acyl, alkyl and heterocyclic unsymmetricalsubstituted imidazolines. After screening a small number of Lewis acidsit was found that TMSC1 (trimethylsilylchloride) promotes thecondensation of azlactones and imines to afford imidazolines in goodyields as single diastereometers (Scheme 1).

Acyl chlorides (RCOCl) where R is chiral can be used to obtain a singleenantiomer. The azlactones were prepared from different N-acyl-α-aminoacids followed by EDCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride) mediated dehydration to provide the pure azlactones inhigh yields (Schunk et al., Organic letters 2: 907-910 (2000); Sain etal., Heterocycles 23: 1611-1614 (1985)). The cycloaddition reactionswith the imines proceeded well at slightly elevated temperatures (forexample 40° C.) to provide the high substituted imidazolines in goodyields. The absence of trimethylsilyl chloride resulted in the formationof β-lactams, presumably via a ketene intermediate (Peddibhotla et al.,Highly Diastereoselective Multicomponent Synthesis of UnsymmetricalImidazolines, Organic Letters 4: 3533-3535 (2002)). Only the transdiastereomers of the imidazolines were observed in most of thesereactions as determined by NOE experiments and X-ray crystallography.The diastereoselective multicomponent one-pot synthesis provided a widerange of aryl, acyl, alkyl and heterocyclic substituted imidazolines inexcellent yields (Table 1).

TABLE 1 Preparation of imidazolines 1-10 com- Yield pound R₁ R₂ R₃ R₄ R₅(%) 1 Phenyl Methyl Phenyl Benzyl H 75 2 Phenyl Methyl 4-methoxy- BenzylH 78 phenyl 3 Phenyl Methyl Phenyl 4-Fluorophenyl H 74 4 Phenyl PhenylPhenyl Benzyl H 65 5 Phenyl 1H-indol- Phenyl Benzyl H 68 3-yl- methyl 6Phenyl methyl pyridin-4-yl Benzyl H 76   7^(a) Phenyl Methyl Phenyl H H70 8 Phenyl Methyl Ethoxy- H H 72 carbonyl   9^(b) Phenyl Methylpyridin-4-yl Benzyl Et 76 10^(c) Phenyl Methyl Phenyl Benzyl Et 75^(a)After hydrogenation (10% Pd/C, H₂ 1 atm) of compound 1. ^(b)Afteresterification (SOCl2, EtOH) of compound 6. ^(c)After esterification(SOCl2, EtOH) of compound 1.

While the complete mechanistic detail of this process is still underinvestigation, the reaction does not seem to proceed by activation ofthe carbonyl oxygen of the oxazolone by trimethylsilyl chloride, in turncausing ring-opening to the intermediate nitrilium ion as initiallyexpected (Ivanova, G. G., Tetrahedron 48 (1992)). Carrying out thecondensation in presence of slight excess of triethylamine halted thereaction altogether suggesting that acidic conditions were required. Inaddition, the addition of Lewis acids such as TiCl₄ or BF₃. OEt₂ did notresult in any product formation. In the light of these findings, it isproposed that the reaction probably proceeds by 1,3-dipolar type ofcycloaddition. Steric repulsion between the R₂ and R₃ moieties duringthe cycloaddition can explain the diastereoselectivity (Scheme 2).

In pharmaceutical compositions, the imidazoline is inhibitory at adosage of 1 to 1,000 micrograms per milliliter or gram. It can be usedin a ratio of 1 to 100 or 100 to 1 with other compounds or drugs for thetreatment of autoimmune diseases or anti-rejection compounds or drugs.In a preferred embodiment, one or more of the imidazolines for treatinga patient are provided to the patient at an inhibitory dose in apharmaceutically acceptable carrier. As such, the imidazolines areprocessed with pharmaceutical carrier substances by methods well knownin the art such as by means of conventional mixing, granulating,coating, suspending and encapsulating methods, into the customarypreparations for oral or rectal administration. Thus, imidazolinepreparations for oral application can be obtained by combining one ormore of the imidizolines with solid pharmaceutical carriers; optionallygranulating the resulting mixture; and processing the mixture orgranulate, if desired and/or optionally after the addition of suitableauxiliaries, into the form of tablets or dragee cores.

Suitable pharmaceutical carriers for solid preparations are, inparticular, fillers such as sugar, for example, lactose, saccharose,mannitol or sorbitol, cellulose preparations and/or calcium phosphates,for example, tricalcium phosphate or calcium hydrogen phosphate; alsobinding agents, such as starch paste, with the use, for example, ofmaize, wheat, rice or potato starch, gelatine, tragacanth, methylcellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl celluloseand/or polyvinylpyrrolidone, esters of polyacrylates orpolymethacrylates with partially free functional groups; and/or, ifrequired, effervescent agents, such as the above-mentioned starches,also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, oralginic acid or a salt thereof, such as sodium alginate. Auxiliaries areprimarily flow-regulating agents and lubricating agents, for example,silicic acid, talcum, stearic acid or salts thereof, such as magnesiumstearate or calcium stearate. Dragee cores are provided with suitablecoatings, optionally resistant to gastric juices, whereby there areused, inter alia, concentrated sugar solutions optionally containing gumarabic, talcum, polyvinylpyrrolidone, and/or titanium dioxide, lacquersolutions in aqueous solvents or, for producing coatings resistant tostomach juices, solutions of esters of polyacrylates orpolymethacrylates having partially free functional groups, or ofsuitable cellulose preparations such as acetylcellulose phthalate orhydroxypropyl-methylcellulose phthalate, with or without suitablesofteners such as phthalic acid ester or triacetin. Dyestuffs orpigments may be added to the tablets or dragee coatings, for example foridentification or marking of the various doses of active ingredient.

Imidazoline preparations comprising one or more of the imidizolineswhich can be administered orally further include hard gelatine capsules,as well as hard or soft closed capsules made from gelatine and, ifrequired, a softener such as glycerin or sorbitol. The hard gelatinecapsules can contain one or more of the imidazolines in the form of agranulate, for example in admixture with fillers such as maize starch,optionally granulated wheat starch, binders or lubricants such astalcum, magnesium stearate or colloidal silicic acid, and optionallystabilizers. In closed capsules, the one or more of the imidazolines isin the form of a powder or granulate; or it is preferably present in theform of a suspension in suitable solvent, whereby for stabilizing thesuspensions there can be added, for example, glycerin monostearate.

Other imidazoline preparations to be administered orally are, forexample, aqueous suspensions prepared in the usual manner, whichsuspensions contain the one or more of the imidizolines in the suspendedform and at a concentration rendering a single dose sufficient. Theaqueous suspensions either contain at most small amounts of stabilizersand/or flavoring substances, for example, sweetening agents such assaccharin-sodium, or as syrups contain a certain amount of sugar and/orsorbitol or similar substances. Also suitable are, for example,concentrates or concentrated suspensions for the preparation of shakes.Such concentrates can also be packed in single-dose amounts.

Suitable imidazoline preparations for rectal administration are, forexample, suppositories consisting of a mixture of one or more of theimidazolines with a suppository foundation substance. Such substancesare, in particular, natural or synthetic triglyceride mixtures. Alsosuitable are gelatine rectal capsules consisting of a suspension of theone or more of the imidazolines in a foundation substance. Suitablefoundation substances are, for example, liquid triglycerides, of higheror, in particular, medium saturated fatty acids.

Likewise of particular interest are preparations containing the finelyground one or more of the imidazolines, preferably that having a medianof particle size of 5 μm or less, in admixture with a starch, especiallywith maize starch or wheat starch, also, for example, with potato starchor rice starch. They are produced preferably by means of a brief mixingin a high-speed mixer having a propeller-like, sharp-edged stirringdevice, for example with a mixing time of between 3 and 10 minutes, andin the case of larger amounts of constituents with cooling if necessary.In this mixing process, the particles of the one or more of theimidazolines are uniformly deposited, with a continuing reduction of thesize of some particles, onto the starch particles. The mixturesmentioned can be processed with the customary, for example, theaforementioned, auxiliaries into the form of solid dosage units; i.e.,pressed for example into the form of tablets or dragees or filled intocapsules. They can however also be used directly, or after the additionof auxiliaries, for example, pharmaceutically acceptable wetting agentsand distributing agents, such as esters of polyoxyethylene sorbitanswith higher fatty acids or sodium lauryl sulphate, and/or flavoringsubstances, as concentrates for the preparation of aqueous suspensions,for example, with about 5- to 20-fold amount of water. Instead ofcombining the imidazoline/starch mixture with a surface-active substanceor with other auxiliaries, these substances may also be added to thewater used to prepare the suspension. The concentrates for producingsuspensions, consisting of the one or more of the imidazoline/starchmixtures and optionally auxiliaries, can be packed in single-doseamounts, if required in an airtight and moisture-proof manner.

In addition, the one or more imidazolines can be administered to apatient intraperitoneally, intranasally, subcutaneously, orintravenously. In general, for intraperitoneal, intranasal,subcutaneous, or intravenous administration, one or more of theimidazolines are provided by dissolving, suspending or emulsifying themin an aqueous or nonaqueous solvent, such as vegetable or other similaroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives. Preferably, the one or moreimidazolines are provided in a composition acceptable forintraperitoneal, subcutaneous, or intravenous use in warm-bloodedanimals or humans. For example, such compositions can comprise aphysiologically acceptable solution such as a buffered phosphate saltsolution as a carrier for the one or more anthraquinones. Preferably,the solution is at a physiological pH. In particular embodiments, thecomposition is injected directly into the patient perfused through thetumor by intravenous administration.

Preparations according to the present invention comprise one or more ofthe imidazolines at a concentration suitable for administration towarm-blooded animals or humans which concentration is, depending on themode of administration, between about 0.3% and 95%, preferably betweenabout 2.5% and 90%. In the case of suspensions, the concentration isusually not higher than 30%, preferably about 2.5%; and conversely inthe case of tablets, dragees and capsules with the one or more of theimidazolines, the concentration is preferably not lower than about 0.3%,in order to ensure an easy ingestion of the required doses of the one ormore imidazolines. The treatment of patients with the preparationscomprising one or more of the imidazolines is carried out preferably byone or more administrations of a dose of the one or more imidazolinewhich over time is sufficient to substantially inhibit NF-κB. Ifrequired, the doses can be administered daily or divided into severalpartial doses which are administered at intervals of several hours. Inparticular cases, the preparations can be used in conjunction with orfollowing one or more other therapies such as radiation or chemotherapy.The administered dose of the one or more imidazolines is dependent bothon the patient (species of warm-blooded animal or human) to be treated,the general condition of the patient to be treated, and on the type ofdisease to be treated or type of organ transplant or skin graft.

The present invention is useful as an immune suppressant for inhibitingautoimmune diseases and rejection of organ and skin transplants becauseNF-κB activation plays a significant role in immune disorders (Ghosh etal., Ann. Rev. Immunol. 16: 225-260 (1998)). Activation of the NF-κBresults in the active transcription of a great variety of genes encodingmany immunologically relevant proteins (Baeuerle and Henkel, Ann. Rev.Immunol. 12: 141-17 (1994); Daelemans et al., Antivir. Chem. Chemother.10:1-14 (1999)). In the case of the human immunodeficiency virus (HIV)infection results in NF-κB activation, which results in regular viralpersistence (Rabson et al., Adv. Pharmacol. 48: 161-207 (2000); Pati etal., J. Virol. 77: 5759-5773 (2003); Quivy et al., J. Virol. 76:11091-11103 (2002); Amini et al., Oncogene 21: 5797-5803 (2002); Takadaet al., J. Virol. 76: 8019-8030 (2002); Chen-Park et al., J. Biol. Chem.277: 24701-24708 (2002); Ballard, Immunol. Res. 23: 157-166 (2001);Baldwin, J. Clin. Invest. 107: 3-6 (2001); Calzado et al., Clin. Exp.Immunol. 120: 317-323 (2000); Roland et al., DNA Cell Biol 18: 819-828(1999); Boykins et al., J. Immunol. 163: 15-20 (1999); Asin et al., J.Virol. 73: 3893-3903 (1999); Sato et al., AIDS Res. Hum. Retroviruses14: 293-29 (1998)). HIV-1 replication is regulated through an variety ofviral regular proteins as well as cellular transcription factors (inparticular NF-κB) that interact with the viral long terminal repeat(LTR) (Asin et al., J. Virol. 73: 3893-3903 (1999)). HIV-1 is able toenter a latent state in which the integrated provirus remainstranscriptionally silent. The ability to continue to infect cellslatently aids the virus to establish persistent infections and avoid thehost immune system. The latent virus can establish large reservoirs ofgenetic variants in T-cells residing in lymphoid tissue. In addition, arecent study implicates NF-κB with the reactivation of latent HIV inT-cells in patents undergoing antiviral therapy (Finzi et al., Science278: 1295-1300 (1997)).

The present invention is useful for treating inflammation disordersbecause NF-κB activation plays a significant role in inflammationdisorders. NF-κB is activated by TNF and other pro-inflammatorycytokines. Inhibition of NF-κB activation by non-toxic inhibitors could,therefore, have clinical use in the treatment of many inflammatorydisordersrheumatoid arthritis, inflammatory bowel disease, asthma,chronic obstructive pulmonary disease (COPD) osteoarthritis,osteoporosis and fibrotic diseases. Related information on this can befound in (Feldmann et al., Ann. Rheum. Dis. 61: Suppl 2, ii13-18 (2002);Gerard and Rollins, Nat. Immunol. 2: 108-115 (2001); Hart et al., Am. J.Respir. Crit. Care Med. 158: 1585-1592 (1998); Lee and Burckart, J.Clin. Pharmacol. 38: 981-99 (1998); Makarov, Arthritis Res. 3: 200-206(2001); Manna et al., J. Immunol. 163: 6800-6809 (1999); Miagkov et al.,Proc. Natl. Acad. Sci. USA 95: 13859-13864 (1998); Miossec, Cell. Mol.Biol. (Noisy-1e-grand) 47: 675-678 (2001); Roshak et al., Curr. Opin.Pharmacol. 2: 316-321 (2002); Tak and Firestein, J. Clin. Invest. 107:7-11 (2001); Taylor, Mol. Biotechnol. 19: 153-168 (2001); Yamamoto andGaynor, J. Clin. Invest. 107: 135-142 (2001); Zhang and Ghosh, J.Endotoxin Res. 6: 453-457 (2000)).

Models for demonstrating the compounds disclosed herein inhibitoryeffect on inflammation include the following.

Septic Shock model: Ref. Journal of Clinical Investigation 100: 972-985(1997). Role of NF-κB in the mortality of Sepsis. Animal model: FemaleBALB/c mice, aged 10-12 wk, 18-20 g were injected intraperitoneally witha mixture of E. coli LPS (Sigma), 1.75 μg in 0.1 mL sterile PBS, pH 7.4)and D-galactosamine (Sigma, 15 mg in 0.1 mL sterile PBS), in order tosensitize them to the lethal effects of LPS (see also, Proc. Natl. Acad.Sci. USA 76: 5939-5943 (1979) and J. Exp. Med. 165, 657-663 (1987).Mortalilty was monitored after 4, 8, 12, 16, 20, and 24 hours.

Inflammation model: Ear edema using PMA as described by Chang, Eur. J.Pharmacol. 142: 197-205 (1987). 20 μL of imidazoline (variousconcentrations), dexamethasone (40 μg/ear) or vehicle (DMSO:Ethanol;25:75 v/v) was applied topically to the right ear of mice 30 minutesbefore and 30 minutes after the application of 20 μL of PMA (5 μg/ear)dissolved in ethanol. Ear swelling was measured 6 hours after PMAapplication using a microgauge and expressed as the mean difference inthickness between the treated (right) and untreated (left) ears. A valueof p<0.05 was considered statistically significant.

The following examples are intended to promote a further understandingof the present invention.

Examples 1-20 Experimental Section

Dl-(3S,4S)-1-Benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid SP-1-61 (1) was made as follows.

A solution of benzaldehyde (0.06 g, 0.57 mmol), benzylamine (0.061 g,0.57 mmol) in dry dichloromethane (15 mL) was refluxed under nitrogenfor 2 hours. 2-Phenyl-4-methyl-4H-oxazolin-5-one (0.1 g, 0.57 mmol) andchlorotrimethylsilane (0.08 g, 0.74 mmol) were added and the mixture wasrefluxed under nitrogen for 6 hours and then stirred overnight at roomtemperature. The reaction mixture was evaporated to dryness undervacuum. The product was precipitated out as a white solid using 1:1dichloromethane/hexanes mixture (0.155 g, 74%). ¹H NMR (300 MHz)(DMSO-d₆): δ 1.8 (3H, s), 4.05 (1H, d, J=15 Hz), 4.95 (1H, d, J=14.8Hz), 5.05 (1H, s), 7.05 (2H, s), 7.25-7.54 (8H, m), 7.74 (2H, t, J=7.2Hz), 7.83 (1H, t, J=6.9 Hz), 8.0 (2H, d, J=8.4 Hz); ¹³C NMR (75 MHz)(DMSO-d₆): δ 25.2, 48.8, 70.4, 73.3, 122.3, 127.8, 128.3, 128.5, 128.9,129.1, 129.3, 129.6, 129.7, 132.3, 133.2, 134, 166.1, 169.5; IR (neat):3350 cm⁻¹, 1738 cm¹; HRMS (EI): calculated for C₂₄H₂₂N₂O₂ [M-H]⁺369.1603. found [M-H]⁺ 369.1610; M.P.: decomposes at 185-190° C.

Dl-(3S,4S)-1-Benzyl-5-(4-methoxyphenyl)-4-methyl-2-phenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid SP-1-63 (2) was made as follows.

A solution of p-anisaldehyde (0.077 g, 0.57 mmol), benzylamine (0.061 g,0.57 mmol) in dry dichloromethane (15 mL) was refluxed under nitrogenfor 2 hours. 2-Phenyl-4-methyl-4H-oxazolin-5-one (0.1 g, 0.57 mmol) andchlorotrimethylsilane (0.08 g, 0.74 mmol) were added and the mixture wasrefluxed under nitrogen for 6 hours and then stirred overnight at roomtemperature. The reaction mixture was evaporated to dryness undervacuum. The product was precipitated out as a white solid using 1:1dichloromethane/hexanes mixture (0.180 g, 78%). ¹H NMR (300 MHz)(CDCl₃+2 drops DMSO-d₆): δ 1.8 (3H, s), 3.8 (3H, s), 3.95 (1H, d, J=15.3Hz), 4.5 (1H, s), 4.9 (1H, d, J=15 Hz), 6.83-6.92 (4H, m), 7.08-7.19(3H, m), 7.3-7.4 (3H) dd, J₁=5.1 Hz, J₂=1.8 Hz), 7.54-7.62 (2H, t, J=7.2Hz), 762-7.68 (1H, t, J=7.2 Hz), 7.9 (2H, d, J=6.9 Hz); ¹³C NMR (75 MHz)(CD₃OD): δ 25.3, 48.8, 55.6, 70.9, 74.1, 115.2, 122.2, 123, 125.5,127.9, 128.4, 129.2, 129.3, 129.6, 129.9, 132.8, 134.2, 161.1, 166.3,168.4; IR (neat): 3388 cm¹ 1738 cm⁻¹; HRMS (EI): calculated forC₂₅H₂₄N₂O₃ [M-H]⁺ 397.1709. found [M-H]⁺ 399.1717; M.P.: decomposes at205-208° C.

Dl-(3S,4S)-1-(4-Fluorophenyl)-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid SP-1-101 (3) was made as follows.

A solution of benzaldehyde (0.060 g, 0.57 mmol), 4-fluoroaniline (0.063g, 0.57 mmol) in dry dichloromethane (15 mL) was refluxed under nitrogenfor 2 hours. 2-Phenyl-4-methyl-4H-oxazolin-5-one (0.1 g, 0.57 mmol) andchlorotrimethylsilane (0.08 g, 0.74 mmol) were added and the mixture wasrefluxed under nitrogen for 6 hours and then stirred overnight at roomtemperature. The reaction mixture was evaporated to dryness undervacuum. The product was precipitated out as a white solid using 1:1dichloromethane/hexanes mixture (0.160 g, 74%). ¹H NMR (300 MHz)(DMSO-d₆): δ 1.98 (3H, s), 5.98 (1H, s), 7.05-7.65 (14H, m); ¹³C NMR (75MHz) (DMSO-d₆) δ 25.2, 71.2, 77.9, 116.9, 117, 117.1, 117.3, 123, 125.1,125.3, 129.3, 129.4, 129.6, 130.1, 130.3, 130.4, 130.5, 132.5, 133.3,134.5, 160.4, 163.7, 165.3, 170.4; IR (neat): 3450 cm⁻¹, 1744 cm⁻¹. HRMS(EI): calculated for C₂₃H₁₉FN₂O₂ [M-H]⁺ 373.1352. found [M-H]⁺ 373.1359;M.P.: decomposes at 230-232° C.

Dl-(3S,4S)-1-Benzyl-2,4,5-triphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid SP-1-125 (4) was made as follows.

A solution of benzaldehyde (0.6 g, 5.7 mmol), benzylamine (0.61 g, 5.7mmol) in dry dichloromethane (120 mL) was refluxed under nitrogen for 2hours. 2,4-Diphenyl-4H-oxazolin-5-one (1.35 g, 5.7 mmol) andchlorotrimethylsilane (0.8 g, 7.4 mmol) were added and the mixture wasrefluxed under nitrogen for 6 hours and then stirred overnight at roomtemperature. The product was purified by silica-gel columnchromatography with 1:5 ethanol/ethyl acetate to afford 2.1 g of productin 65% yield as an off-white solid. ¹H NMR (300 MHz) (CDCL₃): δ 3.8 (1H,d, J=15.6 Hz), 4.62 (1H, d, J=15.6 Hz), 4.98 (1H, s), 6.58 (2H, d, J=8.1Hz), 7.05-7.65 (16H, m), 7.9 (2H, d, J=7.2 Hz); ¹³C NMR (75 MHz) (CDCl₆)δ 29.7, 48.3, 75.6, 79.1, 123.1, 125.7, 126.7, 127.3, 127.4, 127.9,128.1, 128.2, 128.8, 128.9, 129, 129.3, 132.9, 133.8, 136, 143.1, 164.8,168.1; IR (neat): 3400 cm⁻¹ (very broad), 1738 cm⁻¹; HRMS (EI):calculated for C₂₉H₂₄N₂O₂ [(M-H)CO₂]⁺ 387.1526 and observed [M-H)—CO₂]⁺387.1539; M. P.: decomposes at 153-155° C.

Dl-(3S,4S)-1-Benzyl-4-(1H-indol-3-ylmethyl)-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid SP-1-128 (5) was made as follows.

A solution of benzaldehyde (0.6 g, 5.7 mmol), benzylamine (0.61 g, 5.7mmol) in dry dichloromethane (120 mL) was refluxed under nitrogen for 2hours. 4-(1H-Indol-3-ylmethyl)-2-phenyl-4H-oxazol-5-one (1.65 g, 5.7mmol) and chlorotrimethylsilane (0.8 g, 7.4 mmol) were added and themixture was refluxed under nitrogen for 6 hours and then stirredovernight at room temperature. The product was purified by silica-gelcolumn chromatography with 1:5 ethanol/ethyl acetate to afford 3.1 g ofproduct in 68% yield as an off-white solid. ¹H NMR (300 MHz) (DMSO-d₆):δ 3.95 (1H, d, J=16.2 Hz), 4.6 (1H, d, J=16.2 Hz), 5.25 (1H, s), 6.1(2H, d, J=7.8 Hz), 6.9-7.3 (5H, m), 7.3-8.0 (15H, m), ¹³C NMR (75 MHz)(DMSO-d₆) δ 169.6, 166, 136.5, 133.7, 132.5, 132.3, 129.7, 129.4, 128.9,128.7, 128.6, 127.9, 127.8, 126.7, 126.6, 122.7, 121.4, 119, 111, 105.8,74.4, 70.4, 48.5, 32.3; IR (neat): 3420 cm⁻¹ (very broad), 1741 cm¹;HRMS (EI); calculated for C₃₂H₂₇N₃O₂ [M-H]⁺ 484.2025 and observed [M-H]⁺484.2011; M.P.: decomposes at >250° C.

Dl-(3S,4S)-1-Benzyl-4-methyl-2-phenyl-5-pyridin-4yl-4,5-dihydro-1H-imidazole-4-carboxylicacid SP-1-150 (6) was made as follows.

A solution of pyridin-4-carboxalaldehyde (0.061 g, 0.57 mmol),benzylamine (0.061 g, 0.57 mmol) in dry dichloromethane (15 mL) wasrefluxed under nitrogen for 2 hours. 2-Phenyl-4-methyl 4H-oxazolin-5-one(0.1 g, 0.57 mmol) and chlorotrimethylsilane (0.08 g, 0.74 mmol) wereadded and the mixture was refluxed under nitrogen for 6 hours and thenstirred overnight at room temperature. The reaction mixture wasevaporated to dryness under vacuum. The product was isolated using 4:1ethyl acetate/methanol as an off-white solid (0.161 g, 76%). ¹H NMR (300MHz) (DMSO-d₆): δ 1.8 (3H, s), 4.24 (1H, d, J=15.9 Hz), 4.9 (1H, d,J=14.8 Hz), 5.15 (1H, s), 7.0-7.15 (2H, m), 7.25-7.35 (3H, m), 7.45-7.5(2H, m), 7.7-7.9 (3H, m), 7.95-8.05 (2H, m), 8.6-8.7 (2H, m); ¹³C NMR(75 MHz) (DMSO-d₆) δ 25.1, 49.1, 70.6, 71.7, 122.1, 123, 127.9, 128.4,128.8, 129.2, 129.4, 132.8, 133.9, 141.4, 149.8, 166.5, 169.05; IR(neat); 3400 cm⁻¹, 1746 cm¹; HRMS (EI): calculated for C₂₃H₂₁N₃O₂ [M-H]⁺370.1556. found [M-H]⁺ 370.1556; M.P.: decomposes at 185-190° C.

Dl (3S,4S)-4-Methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid: 16/17 [JK1-1-135] (7) was made as follows.

To a well-stirred suspension of imidazoline-4-carboxylic acid 10 (0.1gm, 0.27 mmol) and cyclohexene (0.1 mL, 1.25 mmol) in dry THF (30 mL)added 10% Pd/C (45 mg, 0.06 mmol). The suspension was refluxed for 36hours. The reaction mixture cooled to room temperature and ethanol (10mL) was added. The mixture was filtered through a Celite bed, washedwith ethanol and the filtrate was evaporated under reduced pressure. Thecrude product was purified by column silica-gel chromatography usingethanol, to yield a white solid (0.070 g, 93%). ¹H NMR (300 MHz)(DMSO-d₆) δ 1.76 (s, 3H), 5.34 (s, 1H), 7.34-7.36 (b, 5H), 7.69 (dd,J=8.1, 7.2, 2H), 7.81 (1H, dd, J₁=6.9 Hz and J₂=7.2 Hz), 8.15 (2H, d,J=8.4 Hz); ¹³C NMR (75 MHz) (DMSO-d₆): 25.32, 55.66, 70.79, 72.57,123.12, 128.24, 128.96, 129.42, 129.67, 130.12, 135.42, 136.24, 164.24,170.77; IR (neat) 1734 cm⁻, 1616 cm⁻; MS (EI): calculated for C₁₇H₁₆N₂O₂(m/z) 280.12 observed m/z: 280.1; M.P.: decomposes at 222-224° C.

Dl-(3S,4S)-1-(4-Fluorophenyl)-4-methyl-2-phenyl-4,5-dihydro-1H-imidazole-4,5-dicarboxylicacid 5-ethyl ester SP-1-175 (8) was made as follows.

A solution of ethyl glyoxalate (0.058 g, 0.57 mmol) as 50% solution intoluene (1.03 g/ml), 4-fluoroaniline (0.063 g, 0.57 mmol) in drydichloromethane (15 mL) was refluxed under nitrogen for 2 hours.2-Phenyl-4-methyl-4H-oxazolin-5-one (0.1 g, 0.57 mmol) andchlorotrimethylsilane (0.08 g, 0.74 mmol) were added and the mixture wasrefluxed under nitrogen for 6 hours and then stirred overnight at roomtemperature. The reaction mixture was evaporated to dryness undervacuum. The product was purified by silica-gel column chromatographyusing 4:1 ethyl acetate/methanol, to yield a white solid (0.152 g, 72%).¹H NMR (300 MHz) (CD₃OD): δ 1.2 (3H, t, J=7.2 Hz), 2.03 (3H, s), 4.9(2H, dq, J₁=7.2 Hz, J₂=2.1 Hz), 5.48 (1H, s), 7.1-7.8 (9H, m); ¹³C NMR(75 MHz) (CD₃OD): δ 169.9, 166.2, 164.0, 162.1, 134.4, 131.5, 129.7,129.6, 129.5, 129.3, 121.8, 116.9, 116.7, 75.1, 69.1, 62.9, 24.2, 12.8;1R (neat) 3450 cm⁻¹, 1743 cm¹; HRMS (EI): calculated for C₂₀H₁₉FN₂O₄[M-H]⁺ 369.1251, and observed [M-H]⁺ 369.1255; M.P. decomposes at190-193° C.

Dl-(3S,4S)-1-Benzyl-4-methyl-2-phenyl-5-pyridin-4-yl-4,5-dihydro-1H-imidazole-4-carboxylicacid ethyl ester JK-1-183 (9) was made as follows.

To a well-stirred suspension ofdl-(3S,4S)-1-Benzyl-4-methyl-2-phenyl-5-pyridin-4yl-4,5-dihydro-1H-imidazole-4-carboxylicacid 12 (0.1 g, 0.27 mmol) in dry dichloromethane (30 mL) at 0° C. addeda solution of oxallyl chloride (0.14 g, 1.1 mmol) in dry dichloromethane(5 mL). A solution of DMF (0.001 mL) was added to the reaction mixtureand was stirred at 0° C. for another 2 hours. The dichloromethane wasevaporated under vacuum and the reaction mixture cooled to 0° C. afterwhich absolute ethanol (20 mL) was added. The solution was allowed tostir for an additional 1 hour. The solvent was evaporated under vacuumand the reaction mixture diluted with dichloromethane (30 mL) and washedwith saturated sodium bicarbonate (1-0 mL) and water (10 mL). Theorganic layer was dried over sodium sulfate and was concentrated undervacuum to yield crude product, which was further purified by silica-gelcolumn chromatography using ethyl acetate, to yield a pale yellow oil(0.097 gm, 91%). ¹H NMR (300 MHz) (CDCl₃): δ 0.86 (3H, t, J=7.2 Hz),1.57 (3H, s), 3.64 (2H, q, J=7.2 Hz), 3.83 (1H, d, J=15.3 Hz), 4.27 (1H,s), 4.77 (1H, d, J=15.3, Hz), 6.97 (2H, dd, J₁=7.2 Hz and J₂=2.4 Hz),7.22-7.54 (6H, m), 7.31-7.54 (2H, m), 7.78-7.81 (2H, m), 8.59-8.61 (2H,m). ¹³C NMR (75 MHz) (CDCl₃): δ 13.45, 27.13, 49.47, 60.83, 71.87,77.94, 122.56, 127.79, 127.93, 128.55, 128.70, 130.21, 130.51, 135.82,146.59, 149.75, 166.02, 171.37; IR (neat): 1734 cm⁻¹; MS (EI):calculated for C₂₅H₂₆N₂O₂ (m/z) 399.19 observed m/z: 399.3.

Dl-(3S,4S)-1-Benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid ethyl ester JK-1-186 (10) was made as follows.

To a well-stirred suspension of imidazoline-4-carboxylic acid 10 (0.1gm, 0.27 mmol) in dry methylene chloride (30 mL) at 0° C. added asolution of oxallyl chloride (0.14 g, 1.1 mmol) in dry dichloromethane(5 mL). A solution of DMF (0.001 mL) in dry dichloromethane (1 mL) wasadded to the reaction mixture and was stirred at 0° C. for another 2hours. The dichloromethane was evaporated under vacuum and the reactionmixture cooled to 0° C. after which absolute ethanol (20 mL) was added.The solution was allowed to stir for an additional 1 hour. The solventwas evaporated under vacuum and the reaction mixture diluted withdichloromethane (30 mL) and washed with saturated sodium bicarbonate (10mL) and water (10 mL). The organic layer was dried over sodium sulfateand was concentrated under vacuum to yield crude product, which wasfurther purified by silica-gel column chromatography using ethylacetate, to yield colorless oil (0.095 gm, 89%). ¹H NMR (300 MHz,CDCl₃): δ 0.84 (3H, t, J=7.2 Hz), 1.57 (3H, s), 3.60 (2H, q, J=7.2 Hz),3.85 (1H, d, J=15.3 Hz), 4.32 (1H, s), 4.74 (1H, d, J=15.3 Hz), 6.98(2H, dd, J₁=6.9 Hz and J₂=2.1 Hz), 7.27-7.35 (m, 8H), 7.49-7.51 (2H, m),7.76-7.79 (2H, m); ¹³C NMR (75 MHz, CDCl₃): δ 13.80, 27.13, 49.12,60.06, 71.31, 127.98, 128.03, 128.12, 128.67, 129.02, 129.11, 130.96,136.40, 136.80, 166.11, 171.78; IR (neat); 1730 cm⁻¹, 1495 cm⁻¹; MS(EI): calculated for C₂₆H₂₆N₂O₂ (m/z) 398.2 observed m/z=398.9.

Dl-(3S,4S)-1-Methoxycarbonylmethyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid JK-1-199 (11) were made as follows.

To a well stirred solution of 2-Phenyl-4-methyl-4H-oxazolin-5-one (0.5g, 2.85 mmol) and TMSC1 (0.37 g, 3.42 mmol) in dry dichloromethane (50mL) added a solution of (Benzylidene-amino)-acetic acid methyl ester (0.gm, mmol) in dry methylene chloride (20 mL) and the mixture was refluxedunder nitrogen for 10 hours and then stirred overnight at roomtemperature. The reaction mixture was evaporated to dryness undervacuum. The product was precipitated out as a white solid using a 1:1dichloromethane/hexanes mixture (0.70 g, 70%). ¹H NMR (300 MHz) (CD₃OD):δ 1.99 (3H, (1H, d, J=18.3 Hz), 4.53 (1H, d, J=18.3 Hz), 5.39 (1H, s),7.47-7.50 (5H, m), 7.74-7.87 (5H, m). ¹³C NMR (75 MHz) (CD₃OD): δ 24.23,52.09, 70.83, 75.38, 121.84, 128.26, 128.69, 129.52, 129.75, 131.78,134.02, 167.59, 168.62, 169.19; IR (neat): 3468 cm⁻¹, 1747 cm¹; MS (EI):calculated for C₂₀H₂₀N₂O₄ (m/z) 352.14 observed m/z=353.2; M.P.:decomposes at 215-217° C. s), 3.67 (3H, s), 3.96.

1-Benzyl-5-(4-methoxy-phenyl)-2,4-dimethyl-4,5-dihydro-1H-imidazole-4-carboxylicacid SP-1-189 (12) was made as follows.

A solution of p-anisaldehyde (1.4 g, 10.4 mmol), benzylamine (1.11 g,10.4 mmol) in dry dichloromethane (150 mL) was refluxed under nitrogenfor 2 h. 2,4-dimethyl-4H-oxazolin-5-one SP-1-188 (1f) (1 g, 8.7 mmol)and chlorotrimethylsilane (1.22 g, 11.3 mmol) were added and the mixturewas refluxed under nitrogen for 6 hours and then stirred overnight atroom temperature. The reaction mixture was evaporated to dryness undervacuum. The product was precipitated out as a white solid using a 1:1dichloromethane/hexanes mixture (1.9 g, 65%). ¹H NMR (300 MHz) (CDCl₃):δ 1.13 (3H, s), 2.43 (3H, s), 3.83 (3H, s), 4.17 (1H, d, J=15.9 Hz),4.57 (1H, d, J=15.9 Hz), 5.8 (1H, s) 6.92 (2H, d, J=8 Hz), 7.05 (2H, d,J=8 Hz) 7.2-7.4 (5H, m); ¹³C NMR (75 MHz) (CDCl₃): δ 12.3, 21.9, 47.8,55.2, 70.4, 114.3, 125.2, 126.9, 128.5, 129.3, 133.3, 159.9, 163.2,174.8; IR (neat): 3388 cm⁻¹; 1738 cm⁻¹; HRMS (EI): calculated forC₂₀H₂₂N₂O₃ [M-H]⁺ (m/z)=337.1552. found (m/z) 337.1548.

Dl-(3S,4S)-1-(2-Ethoxycarbonyl-ethyl)-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid JK-1-215 (13) was made as follows.

To a well stirred solution of 2-Phenyl-4-dimethyl-4H-oxazolin-5-one (1.0g, 5.7 mmol) and TMSC1 (1 mL, 6.8 mmol) in dry dichloromethane (80 mL)added a solution of 3-(Benzylidene-amino)-propionic acid ethyl ester(1.4 gm, 6.8 mmol) in dry methylene chloride (60 mL) and the mixture wasrefluxed under nitrogen for 10 hours and then stirred overnight at roomtemperature. The reaction mixture was evaporated to dryness undervacuum. The product was precipitated out as a white solid using a 1:1dichloromethane/hexanes mixture (1.08 g, 51.4%). ¹H NMR (500 MHz)(CD₃OD): δ 1.17 (t, J=7.5, 3H), 1.9 (s, 3H), 2.47-2.52 (m, 1H),2.52-2.71 (m, 1H), 3.34-3.39 (m, 1H), 3.40-4.09 (m, 3H), 5.42 (s, 1H),7.46-7.49 (m, 5H), 7.72-7.87 (m, 5H); ¹³C NMR (100 MHz) (CD₃OD): δ13.35, 24.87, 30.64, 41.64, 61.00, 70.94, 73.51, 122.77, 128.99, 129.21,129.80, 130.10, 132.78, 134.09, 167.32, 169.81, 170.9. IR (neat): 3481cm⁻¹, 1743 cm⁻¹; MS (EI): calculated for C₂₂H₂₄N₂O₄ (m/z) 380.44observed m/z=380.7. M.P.: decomposes at 218-220° C.

Dl-(3S,4S)-1-(1-Methoxycarbonyl-ethyl)-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid JK-1-192 (14) was made as follows.

To a well stirred solution of 2-Phenyl-4-methyl-4H-oxazolin-5-one (0.25g, 1.5 mmol) and TMSC1 (0.23 mL, 1.8 mmol) in dry dichloromethane (50mL) added a solution of 2-(Benzlidene-amino)-propionic acid methyl ester(0.34 gm, 1.8 mmol) in dry methylene chloride (20 mL) and the mixturewas refluxed under nitrogen for 10 hours and then stirred overnight atroom temperature. The reaction mixture was evaporated to dryness undervacuum. The product was precipitated out as a white solid using a 1:1dichloromethane/hexanes mixture (0.340 g, 66%). ¹H NMR (300 MHz)(CD₃OD): δ 1.19 (d, J=6.9, 3H), 2.06 (s, 3H), 3.38 (s, 3H), 4.89 (q,J=6.9, 1H), 544 (s, 1H), 7.43-7.46 (5H, m), 7.75-7.85 (5H, m). ¹³C NMR(75 MHz) (CD₃OD): δ 14.9, 25.6, 52.7, 56.7, 71.9, 72.5, 122.2, 128.8,128.9, 129.6, 130.0, 134.5, 135.8, 169.2, 169.4, 170.4, IR (neat): 3431cm⁻¹, 1740 cm¹; MS (EI): calculated for C₂₁H₂₂N₂O₄ (m/z) 366.4 observedm/z=366.6. M.P.: decomposes at 222-226° C.

1-Benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazol-4-yl)-methanol 14[JK-1-123] (15) was made as follows.

To a well stirred suspension of Lithium aluminum hydride (0.12 gm, 0.3mmol) in dry THF (5 mL) added a solution of1-Benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid (0.1 gm, 0.27 mmol) in dry THF (5 mL) at 0° C. drop wise, stirredat same temperature for 15 min quenched with ice cold saturated ammoniumchloride solution [Caution: Ammonium chloride solution kept at 0° C. forabout 30 minutes; and should be added with extreme care; highlyexothermic reaction and the reaction mixture should be at 0° C.] thenadded about 10 mL of 10% HCl. The reaction mixture diluted with excessof ethyl acetate (100 mL) washed with water (20 mL) dried over anhydroussodium sulfate, filtered through a fluted filter paper and the organiclayer evaporated under reduced pressure to yield the crude product whichwas purified by column chromatography using ethyl acetate. Yield: 79%;viscous oil, IR (neat): 3314, 2928, 1643, 1516; δ H (300 MHz, CD₃Cl₃): δ1.25 (s, 3H), 3.48 (d, J=12, 1H), 3.56 (d, J=11.8, 1H), 3.75 (d, 12.9,1H), 3.87 (s, 1H), 3.94 (d, J=12.9, 1H), 7.28-7.54 (m, 13H), 7.77-7.79(m, 2H), 8.06 (brs, 1H); δ C (75 MHz, CDCl₃): δ 17.25, 51.67, 61.54,66.28, 66.93, 127.266, 127.68, 128.26, 128.56, 128.82, 129.06, 131.77,135.48 138.03, 139.90, 167.91; m/z: 357.2.

1-Benzyl-4-(2-methoxycarbonyl-ethyl)-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid SP-1-201 (16) was made as follows.

solution of benzaldehyde (0.252 g, 2.4 mmol), benzylamine (0.258 g, 2.4mmol) in dry dichloromethane (100 mL) was refluxed under nitrogen for 2hours. 3-(5-Oxo-2-phenyl-4,5-dihydro-oxazol-4-yl)-propionic acid methylester SP-1-182 (1e)(0.5 g, 2 mmol) and chlorotrimethylsilane (0.282 g,2.6 mmol) were added and the mixture was refluxed under nitrogen for 6hours and then stirred overnight at room temperature. The reactionmixture was evaporated to dryness under vacuum. The product wasprecipitated out as a white solid using a 1:1 dichloromethane/hexanesmixture (0.54 g, 60%). ¹H NMR (300 MHz) (CDCl₃): δ 2.05-2.25 (2H, m),2.3-2.5 (2H, m), 3.55 (3H, s), 4.38 (2H, ddd, J₁=4 Hz, J₂=9 Hz, J₃ 25Hz), 4.86 (1H, q, J=3.3), 7.1-7.6 (12H, m), 7.7-7.9 (4H, m); ¹³C NMR (75MHz) (CDCl₃): δ 27.6, 30.1, 43.3, 51.6, 52.7, 127.1, 127.2, 127.3,128.2, 128.3, 131.5, 131.6, 133.3, 137.8, 167.5, 171.4, 173.6; IR(neat): 1734 cm⁻¹, 1653 cm¹; MS (EI): calculated for C₂₄H₂₂N₂O₂ (m/z)442.5. found (m/z) 443.

Dl-(3S,4S)-1-Benzyl-2,4-dimethyl-5-phenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid 15-[JK-1-238] (17) was made as follows.

To a well stirred solution of 2,4-dimethyl-4H-oxazolin-5-one (0.4 g, 3.5mmol) and TMSC1 (0.58 mL, 4.2 mmol) in dry dichloromethane (60 mL) addeda solution of Benzyl-benzylidene-amine (0.82 gm, 4.2 mmol) in drymethylene chloride (40 mL) and the mixture was refluxed under nitrogenfor 10 hours and then stirred overnight at room temperature. Thereaction mixture was evaporated to dryness under vacuum. The product wasprecipitated out as a white solid using a 1:1 dichloromethane/hexanesmixture (0.60 g, 60%). ¹H NMR (300 MHz) (CD₃OD): δ 1.11 (s, 3H), 2.47(s, 3H), 4.17 (d, J=16.2, 1H), 4.63 (q, J=16.2, 1H), 5.84 (s, 1H),7.04-7.07 (m, 2H), 7.27-7.42 (m, 7H). ¹³C NMR (75 MHz) (CD₃OD): δ 12.62,22.12, 48.27, 70.39, 71.25, 127.31, 128.83, 129.28, 129.58, 133.40,133.46, 164.12, 175.19. IR (neat): 3431 cm⁻¹, 1740 cm⁻¹; MS (EI);calculated for C₁₉H₂₀N₂O₂ (m/z) 308.37 observed m/z 308.3, M.P.;decomposes at 232-234° C.

Dl-(3S,4S)-1-Benzyl-2,4-diphenyl-5-pyridin-4-yl-4,5-dihydro-1H-imidazole-4-carboxylicacid SP-1-195 (18) was made as follows.

A solution of pyridin-4-carboxylaldehyde (0.61 g, 0.57 mmol),benzylamine (0.61 g, 5.7 mmol) in dry dichloromethane (120 mL) wasrefluxed under nitrogen for 2 hours. 2,4-Diphenyl-4H-oxazolin-5-one(1.35 g, 5.7 mmol) and chlorotrimethylsilane (0.8 g, 7.4 mmol) wereadded and the mixture was refluxed under nitrogen for 6 hours and thenstirred overnight at room temperature. The product was purified byprecipitation from dichloromethane/ether mixture to afford 1.35 g of theproduct in 55% yield as an off-white solid. ¹H NMR (300 MHz) (CDCl₃): δ4 (1H, d, J=15.6 Hz), 5.0 (1H, d, J=15.6 Hz), 5.38 (1H, s), 7.1-7.65(17H, m), 8.5 (2H, d, J=7.2 Hz); ¹³C NMR (75 MHz) (CDCl₃): δ 45.2, 66.3,75.6, 123.7, 126.5, 126.9, 128.5, 128.6, 128.8, 129.2, 129.3, 131.9,133.5, 134.4, 136.2, 143.4, 149.7, 166.6, 166.9; IR (neat): 3400 cm⁻¹(very broad), 1733 cm⁻¹; MS (EI): calculated for C₂₄H₂₂N₂O₂ (m/z)434.34. found (m/z) 434.2.

Compounds 19 and 20 were made as follows.

Synthesis of1-Benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid (1-phenyl-ethyl)-amide from1-Benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid: JK-1-309

To a well-stirred suspension of1-Benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid (1.0 g, 0.27 mmol) in dry methylene chloride (25 mL),(S)-(−)-1-Phenyl-ethylamine (0.36 g, 29 mmol) was added EDCIHCl (0.57 g,29 mmol), after five minutes added a solution of DMAP (0.35 gm, 29 mmol)in methylene chloride (10 mL) and stirred for 5-6 hours. The reactionmixture was washed with water (2×10 mL), saturated sodium bicarbonate(20 mL), water (20 mL), 2N HCl (20 mL) and then with water (30 mL). Theorganic layer dried over sodium sulfate and evaporated under reducedpressure. The crude product was purified by column silica-gelchromatography using ethyl acetate hexane mixture (1:1). Compound 19:Yield (0.26 g, 40.7%). {[α]_(D)=+41.5°)} ¹H NMR (300 MHz): δ 1.02 (d,J=6.9, 3H), 1.56 (s, 3H), 3.85 (d, J=15.6, 1H), 4.40 (s, 1H), 4.66 (d,J=15.6, 1H), 4.72 (t, J=6.9, 1H), 7.07-7.09 (m, 2H), 7.17-7.55 (m, 16H),7.69-7.73 (m, 2H); ¹³C NMR (75 MHz): 21.39, 27.56, 48.09, 48.73, 72.66,126.52, 127.24, 127.71, 127.99, 128.42, 128.57, 128.67, 128.95, 129.01,129.14, 130.75, 130.82, 137.38, 137.60, 143.29, 165.44, 171.61. Compound20: (0.24 g, 38%). {[α]_(D)=37.7°)} ¹H NMR (300 MHz): δ 1.40 (d, J=7.23H), 1.61 (s, 3H), 3.77 (d, J=15.6, 1H), 4.37 (s, 1H), 4.60 (d, J=15.6,1H), 4.75 (t, J=7.5, 1H), 6.922-7.090 (m, 2H), 7.11-7.22 (m, 13H),7.507-7.529 (m, 3H), 7.651-7.682 (m, 2H): ¹³C NMR (75 MHz): 21.58,28.08, 47.97, 48.59, 72.62, 126.66, 126.99, 127.200, 127.69, 127.96,128.21, 128.51, 128.58, 128.64, 129.13, 129.122, 130.70, 130.83,137.184, 137.22, 143.28, 165.35, 171.62.

Example 21

All compounds were evaluated for their potential anti-inflammatoryactivity by examining the activity of NF-κB in vitro in nuclear extractsusing the procedure from Breton and Charbot-Fletcher (Breton et al., J.Pharmacol. Exp. Ther. 282 459-466 (1997)). Briefly, Human Jurkatleukemia T-cells (clone E6-1; Amer. Type Culture Collection, Manassas,Va.) are grown in RPMI-1640 Media (Gibco-BRL, Bethesda, Md.)supplemented with 10% Fetal Bovine Serum, Penicillin (614 ηg/mL),Streptomycin (10 μg/mL) and Hepes Buffer, pH 7.2 at 37° C., 5% CO₂. TheJurkat cells (1×10⁷ cells/mL) are subsequently treated with variousconcentrations of imidazoline for 30 minutes at 37° C. followed by PMAstimulation (5.0 ng/mL) for an additional 5 hours. Nuclear extracts areincubated for 20 minutes with a double stranded Cy3 labeled NF-κBconsensus oligonucleotide, 5′-AGTTGAGGGGACTTTCCCAGGC-3′ (SEQ ID NO:1) atroom temperature. The crude mixture is loaded on a 5% non-denaturingpolyacrylamide gel prepared in 1× Tris borate/EDTA buffer andelectrophoresed at 200V for 2 hours. After electrophoresis the gel isanalyzed using a phosphorimager (Biorad FX PLUS) for detection of theNF-κB-DNA binding.

Treatment of the cells to the imidazolines exhibited a significantinhibition of nuclear NF-κB activity. FIG. 3 clearly illustrates adecrease of nuclear NF-κB-DNA binding by imidazolines 8-10 (FIG. 3,lanes 5-10).

Cells treated with the imidazolines exhibited a significant inhibitionof nuclear NF-κB activity (FIG. 3). FIG. 3 clearly illustrates asignificant decrease of nuclear NF-κB-DNA binding in the presence 100 nMconcentration of imidazolines 8-10 (FIG. 3, lanes 5-10).

The apparent absence of a slow moving band in lane 5 is indicative ofsignificant (94%) NF-κB inhibition by compound 8 at 1 μM concentrationin Jurkat Leukemia T-cells. Lane 6 indicates 88% inhibition of NF-κB-DNAbinding in the nucleus by 100 ηM concentrations of compound 8.

Example 22

All compounds were tested for their ability to inhibit NF-κB and thecollected data is shown in Table 2. Currently, the most active compoundin the series is the heterocyclic imidazoline 9 which exhibited 88%inhibition of NF-κB at 100 nM concentrations. Preliminary resultsindicate that the imidazolines do not exhibit significant cytotoxicityfor up to 72 hours.

TABLE 2 Inhibition of NF-κB by imidazolines 1-10. compound concentration% inhibition 1 1.0 μM 19% 2 1.0 μM 68% 3 1.0 μM 35% 4 1.0 μM 65% 5 1.0μM  0% 6 0.1 μM 84% 7 0.1 μM 38% 8 0.1 μM 88% 9 0.1 μM 71% 10 0.1 μM 22%The most active compound in this series was compound 8.

IC₅₀ values in Mammalian Jurkat cells Leukemia T cells: IC₅₀ value isdefined as the concentration of compounds at which 50% of theprotein/enzyme is inhibited in cells (Table 3).

TABLE 3 Compound IC₅₀ 1 1.95 μM 2 40 ηM 3 6.5 ηM 4 73 ηM 5 not tested 60.3 μM 7 20 ηM

Example 23

Compounds 4, 6, and 7 were tested for the inhibition of bacteria. Atotal of 9 bacterial strains were screened. The following Gram-negativeand Gram-positive bacteria were included: Staphylococcus aureus,Enterobacter aerogenes, Esherichia coli, Klebsiella pneumonia,Pseudomonas aeruginosa, Serratia marcescens, Bacillus cerius, Bacillussubtillus and micrococcus luteus. Bacterial isolates were removed fromstorage, streaked on to nutrient agar plates and incubated for 18-24hours at 35° C. A working bacterial suspension was prepared bysuspending 3-5 isolated colonies in 5 mL saline solution. The turbidityof this suspension was carefully adjusted photometrically to equal thatof a 0.5 McFarland standard. The zone diameters were determined by astandardized disk diffusion method using cation-supplementedMueller-Hinton agar according to NCCLS guidelines (National Committeefor Clinical Laboratory Standards. Methods for dilution AntimicrobialSusceptibility Tests for Bacteria that Grow Aerobically. Fifth EditionApproved Standard M7-A5. Wayne, Pa.: NCCLS (2000)). Minimum inhibitoryconcentrations (MICs) were considered the lowest concentration that gavea clear zone of inhibition. The inoculated agar plates were incubatedfor 16-20 hours at 35° C. in ambient air. The diameters of the zoneswere read in millimeters. The results are shown in Table 4.

TABLE 4 Microbe MIC Compound 4 Bacillus subtillus 13 mm 50 μg Bacilluscereus 11 mm 50 μg Micrococcus luteus 12 mm 200 μg Staphylococcus aureus12 mm 200 μg Compound 6 Micrococcus luteus 10 mm 200 ηg Compound 7Micrococcus luteus 10 mm 56 ηg

Example 24 Treatment of Imidazoline in RIF-1 Murine Tumor Model

Several of the NF-κB inhibitors (compounds 1, 3, 4, and 6) were testedin animals. Tumor cells were injected, bilaterally, into the backs ofmice. When tumors reached 100 mm³, the mice were treated with anintraperitoneal injection of the compound. Tumor volumes were measured 3times a week until they reached 4 times the size they were on the firsttreatment day. Data is recorded as “Days to 4×’ or ratio of ‘Days to 4×”of the treated over untreated controls.

Combinational treatment of the mice with cis-platin (CDDP) andcamptothecin (CPT) in the presence of compound 4 (1-SP-4-84) exhibitedconsiderable chemopotentiation of cis-platin (FIG. 4A). In addition,this group had 4 of the 8 tumors that remained <4× its volume at day 22of the experiment. No significant chemopotentiation of camptothecin inthe presence of compound 4 was shown.

Compound 6 (1-SP-6-95) exhibited significant chemopotentiation ofcis-platin as well as camptothecin (FIG. 4B). However, chemopotentiationby 6 was not as pronounced as seen with compound 4.

Combinational treatment of the mice with cis-platin (CDDP) andcamptothecin (CPT) in the presence and absence of the imidazolinesindicated that compounds 1 and 3 showed no significant chemopotentiationof either cis-platin or camptothecin (data not shown).

Combinational therapy of compound 4 with cis-platin showed a tumorgrowth delay (in days) of more than 10.26 days as compared to cis-platin(0.82 days) or camptothecin (3.79 days) alone (Table 5). In addition,half of the tumors in this RIF-1 murine model did not reach the 4× tumorvolume cut-off point at day 22 days when exposed to combinationaltreatment with compound 4.

TABLE 5 Antitumor efficacy of imidazolines as measured by the RIF-1murine model of tumor growth delay. CGX-E060 # Dose Days to 4x DaysTreatment of Tumors Route (mg/kg) (Ave ± SE) T/C Median Delay Untreated10  — — 7.3 ± 0.6 0.0 7.0 0.00 Cis-platin 8 IP 4 8.1 ± 0.4 1.1 7.8 0.82Compound 1 8 IP 100 6.5 ± 0.3 0.9 6.4 −0.57 Compound 3 8 IP 100 7.6 ±1.0 1.0 6.8 −0.20 Compound 4 6/8 IP 100 6.4 ± 0.2 0.9 6.5 −0.56 Compound6 8 IP 100 6.6 ± 0.3 0.9 6.6 −0.41 CDDP + 1 8 IP 4/100 8.8 ± 0.5 1.2 9.12.05 CDDP + 3 8 IP 4/100 8.8 ± 0.3 1.2 8.5 1.49 CDDP + 4* 8 IP4/100 >17.1 ± 1.9    >2.3 >17.3 >10.26 CDDP + 6 8 IP 4/100 9.8 ± 0.3 1.310.1 3.09 Camptothecin 8 IP 6 10.3 ± 0.6  1.4 10.8 3.79 CPT + 1 8 IP6/100 10.2 ± 0.4  1.4 10.3 3.27 CPT + 3 8 IP 6/100 8.8 ± 0.6 1.2 8.71.70 CPT + 4 4/8 IP 6/100 10.8 ± 0.4  1.5 11.1 4.07 CPT + 6 8 IP 6/10011.8 ± 0.9  1.6 10.7 3.72 *This group had 4 of 8 tumors <4x at Day 22.Abbreviations: CDDP (cis-platin) and CPT (camptothecin) and IP(intraperitoneal injection).

This data illustrates the efficacy of the imidazolines in thechemopotentiation of commonly used anticancer drugs. Inhibition ofchemoresistance by these novel NF-κB inhibitors (especially compound 4)results in a significant delay of tumor growth as compared to treatmentof the tumors with the anticancer drug alone.

Example 25

As shown in FIGS. 5 and 6, a new class of imidazoline has beensynthesized which are potent inhibitors of NF-κB in T-cells.

We have recently reported a novel highly diastereoselectivemulticomponent one-pot synthesis of substituted imidazolines (FIG. 5)(Peddibhota et al., Org. Lett 4: 3533-3535 (2002)). These low molecularweight scaffolds contain a four-point diversity applicable to alkyl,aryl, acyl, and heterocyclic substitutions. Surprisingly, theutilization of azlactones (or oxazolones) had not yet resulted in anefficient entry into a stereoselective highly diverse class ofimidazoline scaffolds. However, 1,3 dipolar cycloadditions utilizingN-methylated mesoionic oxazolones (or “munchones”) are well known andprovide a general route for the synthesis of pyrroles and imidazoles(Gerard and Rollines, Nat. Immunol. 2: 108-115 (2001); Hart et al., Am.J. Respir. Crit. Care Med. 158: 1885-1592 (1998); Makarov, ArthritisRes. 3: 200-206 (2001)). After screening a small number of Lewis acidswe found that TMSC1 promotes the reaction of oxazolones and imines toafford the imidazolines scaffolds in very good yields as singlediastereomers (FIG. 6). A large library of imidazolines was prepared andseveral members were evaluated for their biological properties. Uponscreening of these agents we found that the imidazolines were potentinhibitors of NF-κB activation. The synthesis of these compounds isdescribed below.

Reactions were carried out in oven-dried glassware under nitrogenatmosphere, unless otherwise noted. All commercial reagents were usedwithout further purification. All solvents were reagent grade. THF wasfreshly distilled from sodium/benzophenone under nitrogen. Toluene,Dichloromethane and TMSC1 were freshly distilled from CaH₂ undernitrogen. All reactions were magnetically stirred and monitored by thinlayer chromatography with Analtech 0.25-mm pre-coated silica gel plates.Column chromatography was carried out on silica gel 60 (230-400 mesh)supplied by EM Science. Yields refer to chromatographically andspectroscopically pure compounds unless otherwise stated. Melting pointswere determined on a MeI-Temp (Laboratory devices) apparatus with amicroscope attachment. Infrared spectra were recorded on a Nicolet IR/42spectrometer. Proton, carbon, and NMR spectra were recorded on a VarianGemini-300 spectrometer or a Varian VXR-500 spectrometer. Chemicalshifts are reported relative to the residue peaks of solvent chloroform(δ 7.24 for ¹H and δ 77.0 for ¹³C) and dimethyl sulfoxide (δ_(—)2.49 for¹H and δ 39.5 for ¹³C). High-resolution mass spectra were obtained atthe Mass Spectrometry Laboratory of the University of South Carolina,Department of Chemistry & Biochemistry with a Micromass VG-70S massspectrometer. Gas chromatography/low-resolution mass spectra wererecorded on a Hewlet-Packard 5890 Series II gas chromatograph connectedto a TRIO-1 EI mass spectrometer. All chemicals were obtained fromAldrich Chemical Co. and used as received.

TABLE 6 Compound IR (cm⁻¹) ¹H NMR (300 MHz) 33 (2a) 3350, 1738(DMSO-d₆): δ 1.8 (3H, s), 4.05 (1H, d, J = 15 Hz), 4.95 (1H, d, J = 14.8Hz), 5.05 (1H, s), 7.05 (2H, s), 7.25-7.54 (8H, m), 7.74 (2H, t, J = 7.2Hz), 7.83 (1H, t, J = 6.9 Hz), 8.0 (2H, d, J = 8.4 Hz) 34 (2b) 3388,1738 (CDCl₃ + 2 drops DMSO-d₆): δ 1.8 (3H, s), 3.8 (3H, s), 3.95 (1H, d,J = 15.3 Hz), 4.5 (1H, s), 4.9 (1H, d, J = 15 Hz), 6.83- 6.92 (4H, m),7.08-7.19 (3H, m), 7.3-7.4 (3H, dd, J₁ = 5.1 Hz, J₂ = 1.8 Hz), 7.54-7.62 (2H, t, J = 7.2 Hz), 762-7.68 (1H, t, J = 7.2 Hz), 7.9 (2H, d, J =6.9 Hz); 28 (2c) 3450, 1744 (DMSO-d₆): δ 1.98 (3H, s), 5.98 (1H, s),7.05-7.65 (14H, m) 31 (2d) 3400, 1746 (DMSO-d₆): δ 1.8 (3H, s), 4.24(1H, d, J = 15.9 Hz), 4.9 (1H, d, J = 14.8 Hz), 5.15 (1H, s), 7.0-7.15(2H, m), 7.25-7.35 (3H, m), 7.4-7.5 (2H, m), 7.7-7.9 (3H, m), −7.95-8.05 (2H, m), 8.6-8.7 (2H, m) 29 (2e) 3450, 1743 (CD₃OD): δ 1.2 (3H, t,J = 7.2 Hz), 2.03 (3H, s), 4.9 (2H, dq, J1 = 7.2 Hz, J2 = 2.1 Hz), 5.48(1H, s), 7.1-7.8 (9H, m) 40 (2g) 3468, 1747 (CD₃OD): δ 1.99 (3H, (1H, d,J = 18.3 Hz), 4.53 (1H, d, J = 18.3 Hz), 5.39 (1H, s), 7.47-7.50 (5H,m), 7.74-7.87 (5H, m) 42 (2h) 3431, 1740 (CD₃OD): δ 1.19 (d, J = 6.9,3H), 2.06 (s, 3H), 3.38 (s, 3H), 4.89 (q, J = 6.9, 1H0, 5.44 (s, 1H),7.43-7.46 (5H, m), 7.75- 7.85 (5H, m). 41 (2i) 3481, 1743 (CD₃OD): δ1.17 (t, J = 7.5, 3H), 1.9 (s, 3H), 2.47-2.52 (m, 1H), 2.52-2.71 (m,1H), 3.34-3.39 (m, 1H), 3.40-4.09 (m, 3H), 5.42 (s, 1H), 7.46-7.49 (m,5H), 7.72-7.87 (m, 5H). 37 (2j)* 1730, 1595 (CDCl₃): δ 0.84 (3H, t, J =7.2 Hz), 1.57 (3H, s), 3.60 (2H, q, J = 7.2 Hz), 3.85 (1H, d, J = 15.3Hz), 4.32 (1H, s), 4.74 (1H, d, J = 15.3 Hz), 6.98 (2H, dd, J₁ = 6.9 Hzand J₂ = 2.1 Hz), 7.27-7.35 (m, 8H), 7.49-7.51 (2H, m), 7.76-7.79 (2H,m). 38 (2k)* 1734, 1597 (CDCl₃): δ 0.86 (3H, t, J = 7.2 Hz), 1.57 (3H,s), 3.64 (2H, q, J = 7.2 Hz), 3.83 (1H, d, J = 15.3 Hz), 4.27 (1H, s),4.77 (1H, d, J = 15.3 Hz), 6.97 (2H, dd, J₁ = 7.2 Hz and J₂ = 2.4 Hz),7.22-7.54 (6H, m), 7.31-7.54 (2H, m), 7.78-7.81 (2H, m), 8.59-8.61 (2H,m). 45 (21)*

(CDCl₃): δ 1.25 (s, 3H), 3.48 (d, J = 12, 1H), 3.56 (d, J = 11.8, 1H),3.75 (d, 12.9, 1H), 3.87 (s, 1H), 3.94 (d, J = 12.9, 1H), 7.28-7.54 (m,13H), 7.77-7.79 (m, 2H), 8.06 (brs, 1H) 39 (2m)

(DMSO-d₆): δ 1.76 (s, 3H), 5.34 (s, 1H), 7.34-7.36 (b, 5H), 7.69 (dd, J= 8.1, 7.2, 2H), 7.81 (1H, dd, J₁ = 6.9 Hz and J₂ = 7.2 Hz), 8.15 (2H,d, J = 8.4 Hz). 32 (2n) 3400, 1738 (CDCl₃): δ 3.8 (1H, d, J = 15.6 Hz),4.62 (1H, d, J = 15.6 Hz), 4.98 (1H, s), 6.58 (2H, d, J = 8.1 Hz),7.05-7.65 (16H, m), 7.9 (2H, d, J = 7.2 Hz). 35 (2o) 3400, 1733 (CDCl₃):δ 4 (1H, d, J = 15.6 Hz), 5.0 (1H, d, J = 15.6 Hz), 5.38 (1H, s),7.1-7.65 (17H, m), 8.5 (2H, d, J = 7.2 Hz). 30 (2p) 3331, 1736 (CDCl₃):δ 0.84 (3H, t, J = 7.2 Hz), 3.89 (2H, dq, J₁ = 7.2 Hz, J₂ = 3 Hz), 4.73(1H, s), 6.7-6.84 (2H, m), 6.89 (2H, t, J = 9 Hz), 7.34-7.5 (3H, m),7.55 (3H, t, J = 7.5 Hz), 7.65 (2H, t, J = 8.1 Hz), 7.83 (2H, dd, J₁ =8.1 Hz, J₂ = 2.1 Hz), 8.1-8.22 (2H, m) 36 (2q) 3420, 1741 (DMSO-d₆): δ3.95 (1H, d, J = 16.2 Hz), 4.6 (1H, d, J = 16.2 Hz), 5.25 (1H, s), 6.1(2H, d, J = 7.8 Hz)), 6.9-7.3 (5H, m), 7.3-8.0 (15H, m). 44 (2r) 1734,1653 (CDCl₃): δ 2.05-2.25 (2H, m), 2.3-2.5 (2H, m), 3.55 (3H, s), 4.38(2H, ddd, J1 = 4 Hz, J2 = 9 Hz, J3 = 25 Hz), 4.86 (1H, q, J = 3.3),7.1-7.6 (12H, m), 7.7-7.9 (4H, m). 2s 3350, 1704 (DMSO-d₆): δ 3.47 (1H,d, J = 15.6 Hz), 4.31 (1H, d, J = 15.6 Hz), 5.8 (1H, s), 6.4-7.4 (20 H,m) 46 (2t) 3350, 1624 (CDCl₃): δ 1.74 (3H, s), 3.67 (1H, d, J = 15.3Hz), 4.11 (1H, d, J = 14.7 Hz), 4.38 (1H, s), 4.46 (1H, d, J = 14.7 Hz)4.59 (1H, d, J = 15.3 Hz), 6.77 (2H, d, J = 7 Hz), 7.0- 7.6 (13H, m) 47(3t) 3350, 1738 (CDCl₃): δ 1.14 (3H, s), 3.94 (1H, d, J = 15.6 Hz), 4.24(2H, q, J = 8.7 Hz), 4.56 (1H, d, J = 15 Hz), 5.74 (1H, s), 6.65 (2H, d,J = 7.5 Hz), 7.0- 7.4 (13H, m) *derivatives: k and l -ethyl esters;m-alcohol.

TABLE 7 Com- pound ¹³C NMR (75 MHz) 33 (2a) (DMSO-d₆): δ 25.2, 48.8,70.4, 73.3, 122.3, 127.8, 128.3, 128.5, 128.9, 129.1, 129.3, 129.6,129.7, 132.3, 133.2, 134, 166.1, 169.5 34 (2b) (CD₃OD): δ 25.3, 48.8,55.6, 70.9, 74.1, 115.2, 122.2, 123, 125.5, 127.9, 128.4, 129.2, 129.3,129.6, 129.9, 132.8, 134.2, 161.1, 166.3, 168.4 28 (2c) (DMSO-d₆): δ25.2, 71.2, 77.9, 116.9, 117, 117.1, 117.3, 123, 125.1, 125.3, 129.3,129.4, 129.6, 130.1, 130.3, 130.4, 130.5, 132.5, 133.3, 134.5, 160.4,163.7, 165.3, 170.4 31 (2d) (DMSO-d₆): δ 25.1, 49.1, 70.6, 71.7, 122.1,123, 127.9, 128.4, 128.8, 129.2, 129.4, 132.8, 133.9, 141.4, 149.8,166.5, 169.05 29 (2e) (CD₃OD): δ169.9, 166.2, 164.0, 162.1, 134.4,131.5, 129.7, 129.6, 129.5, 129.3, 121.8, 116.9, 116.7, 75.1, 69.1,62.9, 24.2, 12.8 40 (2g) (CD₃OD): δ 24.23, 52.09, 70.83, 75.38, 121.84,128.26, 128.69, 129.52, 129.75, 131.78, 134.02, 167.59, 168.62, 169.1942 (2h) (CD₃OD): δ 14.9, 25.6, 52.7, 56.7, 71.9, 72.5, 122.2, 128.8,128.9, 129.6, 130.0, 134.5, 135.8, 169.2, 169.4, 170.4 41 (2i) (CD₃OD):δ 13.3, 24.8, 30.6, 41.6, 61.0, 70.9, 73.5, 122.7, 128.9, 129.2, 129.8,130.1, 132.7, 134.0, 167.3, 169.8, 170.9 37 (2j)* (CDCl₃): δ 13.80,27.13, 49.12, 60.06, 71.31, 127.98, 128.03, 128.12, 128.67, 129.02,129.11, 130.96, 136.40, 136.80, 166.11, 171.78 38 (2k)* (CDCl₃): δ13.45, 27.13, 49.47, 60.83, 71.87, 77.94, 122.56, 127.79, 127.93,128.55, 128.70, 130.21, 130.51, 135.82, 146.59, 149.75, 166.02, 171.3745 (2l)* (CDCl₃): δ 17.25, 51.67, 61.54, 66.28, 66.93, 127.266, 127.68,128.26, 128.56, 128.82, 129.06, 131.77, 135.48, 138.03, 139.90, 167.9139 (2m) (DMSO-d₆): δ 25.32, 55.66, 70.79, 72.57, 123.12, 128.24, 128.96,129.42, 129.67, 130.12, 135.42, 136.24, 164.24, 170.77 32 (2n) (CDCl₃):δ 29.7, 48.3, 75.6, 79.1, 123.1, 125.7, 126.7, 127.3, 127.4, 127.9,128.1, 128.2, 128.8, 128.9, 129, 129.3, 132.9, 133.8, 136, 143.1, 164.8,168.1 35 (2o) (CDCl₃): δ 45.2, 66.3, 75.6, 123.7, 126.5, 126.9, 128.5,128.6, 128.8, 129.2, 129.3, 131.9, 133.5, 134.4, 136.2, 143.4, 149.7,166.6, 166.9 30 (2p) (CDCl₃): δ 176.8, 169, 162.1, 155.7, 142.1, 142,134.6, 133, 128.6, 128.2, 126.1, 125, 117.4, 117.3, 115.6, 115.3, 76.3,66.1, 61.1, 13.3 36 (2q) (DMSO-d₆): δ 169.6, 166, 136.5, 133.7, 132.5,132.3, 129.7, 129.4, 128.9, 128.7, 128.6, 127.9, 127.8, 126.7, 126.6,122.7, 121.4, 119, 111, 105.8, 74.4, 70.4, 48.5, 32.3 44 (2r) (CDCl₃): δ27.6, 30.1, 43.3, 51.6, 52.7, 127.1, 127.2, 127.3, 128.2, 128.3, 131.5,131.6, 133.3, 137.8, 167.5, 171.4, 173.6 2s (DMSO-d₆): δ 30.7, 47.5,72.1, 100.2, 126.2, 126.6, 126.8, 127.3, 127.7, 127.9, 128.0, 128.5,128.6, 129.0, 131.4, 132.5, 133.4, 136.4, 164.4, 171.6 46 (2t) (CDCl₃):δ 175.1, 164.8, 133.5, 132.8, 132.2, 129.7, 129.2, 129.1, 128.9, 128.6,128.3, 127.6, 71.1, 70.6, 48.1, 32.4, 22.1 47 (3t) (DMSO-d₆ + CD3OD): δ170.6, 165.6, 134.2, 133.5, 132.2, 129.9, 129.8, 129.5, 129.4, 129.3,129, 128.9, 128.7, 128.5, 128.3, 127.9, 73.5, 71.5, 48.6, 38.7, 27.1*derivatives: k and l -ethyl esters; m-alcohol.

The general procedure for synthesizing the above compounds was asfollows.

Synthesis of 2-oxazolin-5-ones was a follows. A solution of benzoylamino acid (2 mmol.) and EDCI.HCl (2 mmol.) in dichloromethane (20 mL)was stirred at 0° C. for 1 hour for racemic compounds, 15 minutes foroptically active compounds. The reaction mixture was washed successivelywith cold (containing ice) water, aqueous NaHCO₃, and water (10 mL each)The solution was dried over anhydrous magnesium sulfate, filtered, andthe solvent evaporated to dryness in vacuo giving the oxazolones assolids or oils.

Synthesis of Imidazoline-4-carboxylic acids was as follows. A solutionof aldehyde (1 mmol) and amine (1 mmol) in dry toluene/drydichloromethane (10 mL) was refluxed under nitrogen for 2 hours. Thesolvent was evaporated under reduced pressure and the imine redissolvedin dichloromethane (10 mL). To the solution was added oxazolone (1 mmol)and chlorotrimethylsilane (1.3 mmol) and the mixture was refluxed undernitrogen for 6 hours and then stirred overnight at room temperature. Theproduct was either precipitated out from 1:1 dichloromethane/hexane orisolated after silica gel chromatography with 4:1 ethylacetate/methanol.

Compound 33 (2a),dl-(4S,5S)-1-Benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. A solution of benzaldehyde (0.06 g,0.57 mmol), benzylamine (0.061 g, 0.57 mmol) in dry dichloromethane (15mL) was refluxed under nitrogen for 2 hours.2-Phenyl-4-methyl-4H-oxazolin-5-one (0.1 g, 0.57 mmol) andchlorotrimethylsilane (0.08 g, 0.74 mmol) were added and the mixture wasrefluxed under nitrogen for 6 hours and then stirred overnight at roomtemperature. The reaction mixture was evaporated to dryness undervacuum. The product was precipitated out as a white solid using a 1:1dichloromethane/hexanes mixture (0.155 g, 74%); HRMS (EI): calculatedfor C₂₄H₂₂N₂O₂ [M-H]⁺ 369.1603. found [M-H]⁺ 369.1610; M. P.: decomposesat 185-190° C.

Compound 34 (2b),dl-(4S,5S)-1-Benzyl-5-(4-methoxyphenyl)-4-methyl-2-phenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. A solution of p-anisaldehyde (0.077 g,0.57 mmol), benzylamine (0.061 g, 0.57 mmol) in dry dichloromethane (15mL) was refluxed under nitrogen for 2 hours.2-Phenyl-4-methyl-4H-oxazolin-5-one (0.1 g, 0.57 mmol) andchlorotrimethylsilane (0.08 g, 0.74 mmol) were added and the mixture wasrefluxed under nitrogen for 6 hours and then stirred overnight at roomtemperature. The reaction mixture was evaporated to dryness undervacuum. The product was precipitated out as a white solid using a 1:1dichloromethane/hexanes mixture (0.180 g, 78%). HRMS (EI): calculatedfor C₂₅H₂₄N₂O₃ [M-H]⁺ 397.1709. found [M-H]⁺ 399.1717; M. P.: decomposesat 205-208° C.

Compound 28 (2c),dl-(4S,5S)-1-(4-Fluorophenyl)-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. A solution of benzaldehyde (0.060 g,0.57 mmol), 4-fluoroaniline (0.063 g, 0.57 mmol) in dry dichloromethane(15 mL) was refluxed under nitrogen for 2 hours.2-Phenyl-4-methyl-4H-oxazolin-5-one (0.1 g, 0.57 mmol) andchlorotrimethylsilane (0.08 g, 0.74 mmol) were added and the mixture wasrefluxed under nitrogen for 6 hours and then stirred overnight at roomtemperature. The reaction mixture was evaporated to dryness undervacuum. The product was precipitated out as a white solid using a 1:1dichloromethane/hexanes mixture (0.160 g, 74%). HRMS (EI): calculatedfor C₂₃H₁₉FN₂O₂ [M-H]⁺ 373.1352. found [M-H]⁺ 373.1359; M. P.:decomposes at 230-232° C.

Compound 31 (2d),dl-(4S,5S)-1-Benzyl-4-methyl-2-phenyl-5-pyridin-4yl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. A solution ofpyridin-4-carboxylaldehyde (0.061 g, 0.57 mmol), benzylamine (0.061 g,0.57 mmol) in dry dichloromethane (15 mL) was refluxed under nitrogenfor 2 hours. 2-Phenyl-4-methyl-4H-oxazolin-5-one (0.1 g, 0.57 mmol) andchlorotrimethylsilane (0.08 g, 0.74 mmol) were added and the mixture wasrefluxed under nitrogen for 6 hours and then stirred overnight at roomtemperature. The reaction mixture was evaporated to dryness undervacuum. The product was isolated using 4:1 ethyl acetate/methanol as anoff-white solid (0.161 g, 76%). HRMS (EI): calculated for C₂₃H₂₁N₃O₂[M-H]⁺ 370.1556. found [M-H]⁺ 370.1556; M. P.: decomposes at 185-190° C.

Compound 29 (2e),dl-(4S,5S)-1-(4-Fluorophenyl)-4-methyl-2-phenyl-4,5-dihydro-1H-imidazole-4,5-dicarboxylicacid 5-ethyl ester was synthesized as follows. A solution of ethylglyoxalate (0.058 g, 0.57 mmol) as 50% solution in toluene (1.03 g/mL),4-fluoroaniline (0.063 g, 0.57 mmol) in dry dichloromethane (15 mL) wasrefluxed under nitrogen for 2 hours. 2-Phenyl-4-methyl-4H-oxazolin-5-one(0.1 g, 0.57 mmol) and chlorotrimethylsilane (0.08 g, 0.74 mmol) wereadded and the mixture was refluxed under nitrogen for 6 hours and thenstirred overnight at room temperature. The reaction mixture wasevaporated to dryness under vacuum. The product was purified bysilica-gel column chromatography using 4:1 ethyl acetate/methanol, toyield a white solid (0.152 g, 72%). HRMS (EI): calculated forC₂₀H₁₉FN₂O₄ [M-H]⁺ 369.1251, and observed [M-H]⁺ 369.1255; M. P.:decomposes at 190-193° C.

Compound 40 (2g),dl-(4S,5S)-1-Methoxycarbonylmethyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. To a well stirred solution of2-Phenyl-4-methyl-4H-oxazolin-5-one (0.5 g, 2.85 mmol) and TMSC1 (0.37g, 3.42 mmol) in dry dichloromethane (50 ml) added a solution of(benzylidene-amino)-acetic acid methyl ester (0. gm, mmol) in drymethylene chloride (20 ml) and the mixture was refluxed under nitrogenfor 10 hours and then stirred overnight at room temperature. Thereaction mixture was evaporated to dryness under vacuum. The product wasprecipitated out as a white solid using a 1:1 dichloromethane/hexanesmixture (0.70 g, 70%). MS (EI): calculated for C₂₀H₂₀N₂O₄ (m/z) 352.14observed m/z=353.2; M. P.: decomposes at 215-217° C.

Compound 42 (2h),dl-(4S,5S)-1-(1-Methoxycarbonyl-ethyl)-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. To a well stirred solution of2-Phenyl-4-methyl-4H-oxazolin-5-one (0.25 g, 1.5 mmol) and TMSC1 (0.23ml, 1.8 mmol) in dry dichloromethane (50 ml) added a solution of2-(Benzylidene-amino)-propionic acid methyl ester (0.34 gm, 1.8 mmol) indry methylene chloride (20 ml) and the mixture was refluxed undernitrogen for 10 hours and then stirred overnight at room temperature.The reaction mixture was evaporated to dryness under vacuum. The productwas precipitated out as a white solid using a 1:1dichloromethane/hexanes mixture (0.340 g, 66%). MS (EI): calculated forC₂₁H₂₂N₂O₄ (m/z) 366.4 observed m/z=366.6. M.P.: decomposes at 222-226°C.

Compound 41 (2i),dl-(4S,5S)-1-(2-Ethoxycarbonyl-ethyl)-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. To a well stirred solution of2-Phenyl-4-dimethyl-4H-oxazolin-5-one (1.0 g, 5.7 mmol) and TMSC1 (1 ml,6.8 mmol) in dry dichloromethane (80 ml) added a solution of3-(benzylidene-amino)-propionic acid ethyl ester (1.4 gm, 6.8 mmol) indry methylene chloride (60 ml) and the mixture was refluxed undernitrogen for 10 hours and then stirred overnight at room temperature.The reaction mixture was evaporated to dryness under vacuum. The productwas precipitated out as a white solid using a 1:1dichloromethane/hexanes mixture (1.08 g, 51.4%). MS (EI): calculated forC₂₂H₂₄N₂O₄ (m/z) 380.44 observed m/z=380.7. M.P.: decomposes at 218-220°C.

Compound 37 (2j),dl-(4S,5S)-1-Benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid ethyl ester was synthesized as follows. To a well-stirredsuspension of imidazoline-4-carboxylic acid 2a (0.1 g, 0.27 mmol) in drymethylene chloride (30 mL) at 0° C. added a solution of oxallyl chloride(0.14 g, 1.1 mmol) in dry dichloromethane (5 mL). A solution of DMF(0.001 mL) in dry dichloromethane (1 mL) was added to the reactionmixture and was stirred at 0° C. for another 2 hours. Thedichloromethane was evaporated under vacuum and the reaction mixturecooled to 0° C. after which absolute ethanol (20 mL) was added. Thesolution was allowed to stir for an additional 1 hour. The solvent wasevaporated under vacuum and the reaction mixture diluted withdichloromethane (30 mL) and washed with saturated sodium bicarbonate (10mL) and water (10 mL). The organic layer was dried over sodium sulfateand was concentrated under vacuum to yield crude product, which wasfurther purified by silica-gel column chromatography using ethylacetate, to yield colorless oil (0.095 gm, 89%). MS (EI): calculated forC₂₆H₂₆N₂O₂ (m/z) 398.2 observed m/z=398.9.

Compound 38 (2k),dl-(4S,5S)-1-Benzyl-4-methyl-2-phenyl-5-pyridin-4yl-4,5-dihydro-1H-imidazole-4-carboxylicacid ethyl ester was synthesized as follows. To a well-stirredsuspension ofdl-(3S,4S)-1-benzyl-4-methyl-2-phenyl-5-pyridin-4yl-4,5-dihydro-1H-imidazole-4-carboxylicacid 2d (0.1 g, 0.27 mmol) in dry dichloromethane (30 mL) at 0° C. addeda solution of oxallyl chloride (0.14 g, 1.1 mmol) in dry dichloromethane(5 mL). A solution of DMF (0.001 mL) in dry dichloromethane (1 mL) wasadded to the reaction mixture and was stirred at 0° C. for another 2hours. The dichloromethane was evaporated under vacuum and the reactionmixture cooled to 0° C. after which absolute ethanol (20 mL) was added.The solution was allowed to stir for an additional 1 hour. The solventwas evaporated under vacuum and the reaction mixture diluted withdichloromethane (30 mL) and washed with saturated sodium bicarbonate (10mL) and water (10 mL). The organic layer was dried over sodium sulfateand was concentrated under vacuum to yield crude product, which wasfurther purified by silica-gel column chromatography using ethylacetate, to yield a pale yellow oil (0.097 gm, 91%). MS (EI): calculatedfor C₂₅H₂₆N₂O₂ (m/z) 399.19 observed m/z: 399.3.

Compound 45 (2l),dl-(4S,5S)-1-Benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazol-4-yl)-methanolwas synthesized as follows. To a well stirred suspension of Lithiumaluminum hydride (0.12 gm, 0.3 mmol) in dry THF (5 ml) added a solutionof 1-benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid (0.1 gm, 0.27 mmol) in dry THF (5 ml) at 0° C. drop wise, stirredat same temperature for 15 minutes quenched with ice cold saturatedammonium chloride solution [Caution: Ammonium chloride solution kept at0° C. for about 30 minutes; and should be added with extreme care;highly exothermic reaction and the reaction mixture should be at 0° C.]then added about 10 ml of 10% HCl. The reaction mixture diluted withexcess of ethyl acetate (100 ml) washed with water (20 ml) dried overanhydrous sodium sulfate, filtered through a fluted filter paper and theorganic layer evaporated under reduced pressure to yield the crudeproduct which was purified by column chromatography using ethyl acetate.yield: 79%; viscous oil, m/z: 357.2.

Compound 39 (2m),dl-(4S,5S)-4-Methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. To a well-stirred suspension ofimidazoline-4-carboxylic acid 2a (0.1 g, 0.27 mmol) and cyclohexene (0.1mL, 1.25 mmol) in dry THF (30 mL) was added 10% Pd/C (45 mg, 0.06 mmol).The suspension was refluxed for 36 hours. The reaction mixture cooled toroom temperature and ethanol (10 mL) was added. The mixture was filteredthrough a Celite bed, washed with ethanol and the filtrate wasevaporated under reduced pressure. The crude product was purified bycolumn silica-gel chromatography using ethanol, to yield a white solid(0.070 g, 93%). MS (EI): calculated for C₁₇H₁₆N₂O₂ (m/z) 280.12 observedm/z: 280.1; M. P.: decomposes at 222-224° C.

Compound 32 (2n),dl-(4S,5S)-1-Benzyl-2,4,5-triphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. A solution of benzaldehyde (0.6 g, 5.7mmol), benzylamine (0.61 g, 5.7 mmol) in dry dichloromethane (120 mL)was refluxed under nitrogen for 2 hours. 2,4-Diphenyl-4H-oxazolin-5-one(1.35 g, 5.7 mmol) and chlorotrimethylsilane (0.8 g, 7.4 mmol) wereadded and the mixture was refluxed under nitrogen for 6 hours and thenstirred overnight at room temperature. The product was purify bysilica-gel column chromatography with 1:5 ethanol/ethyl acetate toafford 2.1 g of the product in 65% yield as an off-white solid. HRMS(EI): calculated for C₂₉H₂₄N₂O₂ [(M-H)—CO₂]+387.1526 and observed[(M-H)—CO₂]+387.1539; M. P.: decomposes at 153-155° C.

Compound 35 (2o),dl-(4S,5S)-1-Benzyl-2,4-diphenyl-5-pyridin-4-yl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. A solution ofpyridin-4-carboxylaldehyde (0.61 g, 0.57 mmol), benzylamine (0.61 g, 5.7mmol) in dry dichloromethane (120 mL) was refluxed under nitrogen for 2hours. 2,4-Diphenyl-4H-oxazolin-5-one (1.35 g, 5.7 mmol) andchlorotrimethylsilane (0.8 g, 7.4 mmol) were added and the mixture wasrefluxed under nitrogen for 6 hours and then stirred overnight at roomtemperature. The product was purified by precipitation fromdichloromethane/ether mixture to afford 1.35 g of the product in 55%yield as an off-white solid. MS (EI): calculated for C₂₄H₂₂N₂O₂ (m/z)434.34. found (m/z) 434.2

Compound 30 (2p),dl-(4S,5S)-1-(4-Fluorophenyl)-2,4-diphenyl-4,5-dihydro-1H-imidazole-4,5-dicarboxylicacid 5-ethyl ester was synthesized as follows. A solution of ethylglyoxalate (0.85 g, 8.3 mmol) as 50% solution in toluene (1.03 g/mL),4-fluoroaniline (0.93 g, 8.3 mmol) in dry dichloromethane (250 mL) wasrefluxed under nitrogen for 2 hours. 2,4-diphenyl-4H-oxazolin-5-one (2g, 8.3 mmol) and chlorotrimethylsilane (1.16, 10.8 mmol) were added andthe mixture was refluxed under nitrogen for 6 hours and then stirredovernight at room temperature. The reaction mixture was evaporated todryness under vacuum.

The product was purified by precipitation with dichloromethane/ether toyield a white solid (2.4 g, 68%). MS (EI): calculated for C₂₅H₂₁FN₂O₄[M]+432.15, and observed [M]⁺ 432.4;

Compound 36 (2q),dl-(4S,5S)-1-Benzyl-4-(1H-indol-3-ylmethyl)-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. A solution of benzaldehyde (0.6 g, 5.7mmol), benzylamine (0.61 g, 5.7 mmol) in dry dichloromethane (120 mL)was refluxed under nitrogen for 2 hours.4-(1H-Indol-3-ylmethyl)-2-phenyl-4H-oxazol-5-one (1.65 g, 5.7 mmol) andchlorotrimethylsilane (0.8 g, 7.4 mmol) were added and the mixture wasrefluxed under nitrogen for 6 hours and then stirred overnight at roomtemperature. The product was purified by silica-gel columnchromatography with 1:5 ethanol/ethyl acetate to afford 3.1 g of productin 68% yield as an off-white solid. HRMS (EI): calculated for C₃₂H₂₇N₃O₂[M-H]⁺ 484.2025 and observed [M-H]⁺ 484.2011; M.P.: decomposes at >250°C.

Compound 44 (2r),dl-(4S,5S)-1-Benzyl-4-(2-methoxycarbonyl-ethyl)-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. A solution of benzaldehyde (0.252 g,2.4 mmol), benzylamine (0.258 g, 2.4 mmol) in dry dichloromethane (100mL) was refluxed under nitrogen for 2 hours.3-(5-Oxo-2-phenyl-4,5-dihydro-oxazol-4-yl)-propionic acid methyl esterSP-1-182 (1e) (0.5 g, 2 mmol) and chlorotrimethylsilane (0.282 g, 2.6mmol) were added and the mixture was refluxed under nitrogen for 6 hoursand then stirred overnight at room temperature. The reaction mixture wasevaporated to dryness under vacuum. The product was precipitated out asa white solid using a 1:1 dichloromethane/hexanes mixture (0.54 g, 60%).MS (EI): calculated for C₂₄H₂₂N₂O₂ (m/z) 442.5. found (m/z) 442.2.

Compound 2s,dl-(4S,5S)-1,2-dibenzyl-4,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid was synthesized as follows. A solution of benzaldehyde (0.6 g, 5.7mmol), benzylamine (0.61 g, 5.7 mmol) in dry dichloromethane (120 mL)was refluxed under nitrogen for 2 hours.2-benzyl-4-phenyl-4H-oxazolin-5-one (1.43 g, 5.7 mmol) andchlorotrimethylsilane (0.8 g, 7.4 mmol) were added and the mixture wasrefluxed under nitrogen for 6 hours and then stirred overnight at roomtemperature. The product was a mixture of diastereomers (3:1 ratio) andwas obtained in 60% yield. The above trans-diastereomer was obtained byrepeated precipitation from methanol/ether. HRMS (EI): calculated forC₃₀H₂₆N₂O₂ [(M-H)—CO₂]⁺ 402.5 and observed [(M-H)—CO₂]⁺ 402.1.

Compound 46 (2t),dl-(4S,5S)-1,2-Dibenzyl-4-methyl-5-phenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid, and Compound 47 (3t),dl-(4S,5R)-1,2-Dibenzyl-4-methyl-5-phenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid were synthesized as follows. A solution of benzaldehyde (1.13 g,10.58 mmol), benzylamine (1.13 g, 10.58 mmol) in dry dichloromethane(250 mL) was refluxed under nitrogen for 2 hours.2-benzyl-4-methyl-4H-oxazolin-5-one (2 g, 10.58 mmol) andchlorotrimethylsilane (1.48 g, 10.58 mmol) were added and the mixturewas refluxed under nitrogen for 6 hours and then stirred overnight atroom temperature. The reaction mixture was evaporated to dryness undervacuum. The product, that precipitated out as a white solid using adichloromethane/ether mixture (0.6 g, 30%), was found to be thecis-isomer (3t). The trans-isomer (2t) then precipitated out from themother liquor. A small amount was then reprecipitated to remove tracesof (3) (0.15 g, 7%) and for characterization.

The compounds and method for synthesis are summarized in Tables 8 and 9.

TABLE 8

% % Yield Ratio Yield R₁ R₂ R₃ R₄ 1 2:3 2 a Ph Me Ph Bn 75 >95:5 74 b PhMe 4-methoxyphenyl Bn 75 >95:5 78 c Ph Me Ph 4-fluorophenyl 75 >95:5 74d Ph Me 4-pyridinyl Bn 75 >95:5 76 e Ph Me —CO₂Et 4-fluorophenyl75 >95:5 72 f Ph Me Ph benzhydryl 75 >95:5 0 g Ph Me Ph —CH₂CO₂Me75 >95:5 70 h Ph Me Ph —CH(CH₃) 75 >95:5 66 CO₂Me i Ph Me Ph —CH₂CH₂ 75 75:25 51 CO₂Et j* Ph Me Ph Bn 75 ethyl 75 ester k* Ph Me 4-pyridinyl Bn75 ethyl 76 ester l* Ph Me Ph Bn 75 alcohol 79 m* Ph Me Ph H 75 >95:5 71*derivatives: k and l—ethyl esters; l—alcohol.

TABLE 9

% % Yield Ratio Yield* R₁ R₂ R₃ R₄ 1 2:3 2 a Ph Me Ph Bn 75 >95:5 74 nPh Ph Ph Bn 70 >95:5 65 o Ph Ph 4- Bn 75 >95:5 55 pyridinyl p Ph Ph—CO₂Et 4- 75 >95:5 68 fluorophenyl q Ph indolyl-3- Ph Bn 80 >95:5 68methyl r Ph —CH₂CH₂ Ph Bn 69 >95:5 60 CO₂Me s Bo Ph Ph Bn 90 75:25 45 tBn Me Ph Bn 76 67:33 N.O u Me Me Ph Bn 60 50:50 N.O N.O.—not optimized

Example 26

The ability of the imidazolines in FIG. 5 to inhibit the nucleartranslocation of NF-κB is described. Inhibition of the nucleartranslocation of the p50/p65 heterodimer of NF-κB was confirmed by ap65-ELISA assay. Cells were treated with the PDTC (positivecontrol-reported to inhibit IκB phosphorylation thus inhibiting NF-κBtranslocation), and imidazoline 32, 30 minutes prior to PMA activation,followed by the isolation of the nuclear extract after a 30 minuteincubation period. PDTC and the imidazolines, but not hymenialdisine(negative control-inhibits NF-κB-DNA binding, but not nucleartranslocation) indicated significant inhibition of NF-κB p50/p65heterodimer translocation at concentrations ranging from 100 ηM-5.0 μM(FIG. 7, for a representative titration of imidazoline 32). Theseresults, therefore, indicate that the imidazoline 32 significantlyinhibits NF-κB translocation at submicromolar concentrations, and wereeven found to be more active than PDTC. Similar results were obtainedfor compounds 28-33 (FIG. 8).

Compounds 28-33 were evaluated for their toxicity in T-cells and werefound to exhibit no significant toxicity. Cell death was measured over a48 hour period and the results shown in Table 10 and a representativegraph of the “most toxic” inhibitor 32 is shown on FIG. 9.

TABLE 10 Compound (1.0 uM) Apoptosis (±S.D.) Cells only 1.00(normalized) 28 1.25 ± 0.18 29 0.95 ± 0.24 30 1.18 ± 0.20 31  1.18 ±0.00^(b) 32  1.98 ± 0.0001

Prior to the combinatorial studies, the toxicity of the compounds wasdetermined in the mouse model. Compounds 28-33 showed no apparenttoxicity to the mice at concentrations up to 100 mg/kg. In short, thedrugs are dissolved in DMSO and 100 μL given to the mice on day 0 byintraperitoneal injection (IP). Initially, doses (5.0 and 50 mg/kg ofcompounds 28, 32, and 33) were used. Since these were well tolerated(FIG. 6A), compounds (29 and 31) were used at 50 and 100 mg/kg (FIG.10B). If weight did not drop at least 10% from the initial weight, thenthe toxicity was not considered significant. Weight and death were thetwo measures of toxicity. No toxicity was observed in these modelsduring a two-week period. Compound 30 was also found to be non-toxic inthe same type of experiment.

The results show that these novel imidazolines are potent inhibitors ofthe transcription factor NF-κB and were found to be non-toxic in T-cellsand animal models. The combination of a non-toxic potent NF-κB inhibitoris useful in the treatment of NF-κB regulated diseases and disorderssuch as inflammatory diseases, certain viral infections, autoimmunediseases, and inhibiting rejection of organ and tissue transplants.

Example 27

This example shows the inhibitory effects of imidazolines 28-33 on NF-kBactivation and its ability to cehmopotentiate cis-platin.

Camptothecin (CPT) is an alkaloid isolated from the extracts of thefruit of Camptotheca acuminata Decaisne and was found to be atopoisomerase inhibitor (Denny, ACS Press: Washington, D.C., 483-500(1995)). Currently, CPT-11 and several water soluble analogues includingtopotecan have successfully past clinical trials in the United States(Wall, Med. Res. Rev. 18: 299-314 (1998)). Camptothecin exhibits itsantitumor activity via the formation of a stable ternary topoisomeraseI-DNA cleavable complex. Stabilization of this cleaved DNA complexinitiates a signaling pathway, ultimately resulting in apoptotic celldeath (Pommier et al., Biochim. Biophys. Acta 1400: 83-105 (1998);Macdonald et al., Comprehensive natural products chemistry;Elsevier-North Holland: Amsterdam, pp 593-614 (1999)).

In addition to the stabilization of the topoisomerase I-DNA cleavablecomplex, camptothecin also activates DNA repair mechanism mediated bythe nuclear transcription factor NF-κB (Boland, Biochem Soc Trans 29:674-678 (2001); Bottero et al., Cancer Res 61: 7785-7791 (2001); Huangetal., J Biol Chem 275: 9501-9509 (2000); Piret and Piette, Nucl. AcidsRes. 24: 4242-4248 (1996)). Activation of NF-κB by DNA damaging agentssuch as the camptothecin (CPT) has been documented in different celllines by several groups (Bottero et al., Cancer Res 61: 7785-7791(2001); Huanget al., J Biol Chem 275: 9501-9509 (2000); Piret andPiette, Nucl. Acids Res. 24: 4242-4248 (1996)). CEM leukemia T-cellswere demonstrated to be sensitive to NF-κB activation by camptothecinand these results were confirmed in our laboratory using an EMSA assay(FIG. 3) (Piret and Piette, Nucl. Acids Res. 24: 4242-4248 (1996)). CEMcells were incubated with and without varying concentrations ofcamptothecin (CPT). Positive controls included PMA/PHA activation (FIG.11, lane 2). Lane 3 included a positive control using PMA/PHA activationfollowed by the treatment of the extract with a NF-κB p65 antibody tounambiguously identify the NF-κB/DNA complex.

As anticipated, treatment of the nuclear extract with the p65 antibodyresulted in a significant decrease in NF-κB/DNA binding (lane 3). As anegative control, cells were left unactivated and nuclear extracts weretreated with the NF-κB consensus sequence, illustrating only a slightbackground level of NF-κB (lane 4). Treatment of the CEM cells withcamptothecin concentrations ranging from 10 μM to 10 nM (FIG. 11, lanes5-8) illustrated a significant amount of NF-κB/DNA binding due to NF-κBactivation.

Inhibition of camptothecin mediated activation of NF-κB by imidazolinesis as follows. Induction of NF-κB activation can proceed via a widerange of signaling pathways (Delhase et al., Science 284: 309-313(1999); Karin, Oncogene 18: 6867-6874 (1999)). Inhibition of NF-κBactivation can proceed via the inhibition of many different pathways(Epinat and Gilmore, Oncogene 18: 6896-6909 (1999)). Modulators of thesepathways may be therefore act as general activation inhibitors, whereasothers may inhibited specific induction pathways (Epinat and Gilmore,Oncogene 18: 6896-6909 (1999)). In order to investigate whether theimidazolines inhibit the specific pathway of camptothecin induced NF-κBactivation, we examined the inhibition of camptothecin induced NF-κBnuclear binding in the presence of the imidazolines.

CEM cells were treated with various concentration of imidazoline 32, 30minutes prior to activation by camptothecin (0.1 μM). As illustrated inFIG. 12, the addition of imidazoline 32 inhibited camptothecin inducedNF-κB nuclear binding in a dose responds manner. Control lanes include:DNA only (lane 1), PMA/PHA activated NF-κB (lane 2), PMA/PHA activatedNF-κB treated with a p65 antibody, which provide a supershift (lane 3)and the unactivated control (lane 4). Activation of NF-κB withcamptothecin provide a strong band indicative of the NF-κB-DNA complex(lane 5). Inhibition of DNA binding in the presence of the non-selectiveNF-κB inhibitor PDTC resulted in a decrease of binding (lane 6). Asimilar decrease of camptothecin induced DNA binding is clearlyillustrated in lanes 7-10, upon treatment of imidazoline 32 ranging from10 μM to 10 nM concentration. Comparison of lane 5 (activated by 0.1 μMCPT) with lane 7 (activated by 0.1 μM CPT+10 μM compound 32) indicates asignificant decrease in NF-κB-DNA complex formation.

Imidazolines 28-33 were evaluated for their ability to enhance theactivity of camptothecin in CEM cells. Induction of apoptosis is thehallmark of most chemotherapeutic agents including camptothecin. Thesystematic disassembly of apoptotic cells is accomplished by activecaspases (Thornberry et al., Nature 356: 768-774 (1992); Nicholson etal., Trends Biochem. Sci. 22: 299-306 (1997)). A standard apoptosisassay is Promega's APO-ONE homogeneous caspase-3/7 assay, which takesadvantage of this caspase activity to directly quantify the induction ofapoptosis in cells (Thornberry et al., Nature 356: 768-774 (1992);Nicholson et al., Trends Biochem. Sci. 22: 299-306 (1997)). According tothe hypothesis that NF-κB inhibition should enhance the activity ofchemotherapeutic agents via the inhibition of anti-apoptotic signalingpathway—thus enhancement of apoptosis—(Boland, Biochem Soc Trans 29:674-678 (2001); Chiao et al., Cancer 95: 1696-1705 (2002)), weinvestigated the effects of the imidazolines using this caspase-3/7assay. This assay quantifies the level of apoptotic cell death inducedby camptothecin with and without the imidazolines and will establish thedirect level of enhancement of apoptosis by camptothecin. Representativecell death graphs are shown in FIGS. 13A-F.

The graphs illustrate two very important biological effects. Theimidazolines appear to be non-toxic (or at least exhibit no significantcytotoxic effects in the CEM cells) as illustrate by the drug(imidazoline) only line (black triangle). The imidazolines appear tosignificantly induce apoptosis when the agents are used in combinationwith the topoisomerase inhibitor, camptothecin (CPT) (line with X versusCPT only line with square). The concentration of camptothecin was keptconstant at 0.1 μM in all experiments and a significant, dose-timeresponse induction of apoptosis was noted upon combinational treatmentwith the imidazolines.

TABLE 11 Fold enhancement of Compound camptothecin (0.1 μM) (1.0 μM)Apoptosis ± S.D. after 48 hours Cells only 1.00 (normalized) 28 1.25 ±(0.18) 29 0.95 ± (0.24) 30 1.18 ± (0.20) 31  1.18 ± (0.00)^(b) 32  1.98± (0.0001) CPT (0.1 μM) 3.17 ± (1.35) 1.00 CPT + 28 4.55 ± (0.83) 1.44CPT + 29 7.32 ± (0.93) 2.26 CPT + 30 7.15 ± (0.11) 2.31 CPT + 31  7.74 ±(0.00)^(b) 2.44 CPT + 32 12.63 ± (0.85)^(a ) 3.98 ^(a)Complete set ofdata shown in FIG. 13A-F. ^(b)Data from single experiment.

One of the most active compounds in the series, thus far, is thetriphenyl substituted benzyl imidazoline 32. The imidazoline 32 at 1.0μM enhanced the camptothecin-induced level of apoptosis 4 fold (seeTable 11 and FIG. 14).

No significant induction of cell death was observed when cells weretreated with only the imidazolines up to 1.0 μM over 48 hours in thisapoptosis (caspase-3) assay as well as tested by cell count up to 10 μMover 72 hours (data not shown). A summary of our results of theinduction of apoptosis by several other imidazolines is depicted inTable 11.

This was determined by the number of apoptotic cell death after 48 hoursafter treatment of CEM cells with 0.1 μM camptothecin (CPT) compared tonumber of dead cells after a combinational treatment of 0.1 μMcamptothecin (CPT) and 1.0 μM imidazoline 32.

In a similar experiment, the imidazoline 32 was found to chemopotentiatecis-platin (FIGS. 15A and 15B). Combination of 0.1 micromolar cis-platinwith 0.1 micromolar 32 was found induce more apoptosis in T-cells (CEMcells) that 1.0 micromolar of cis-platin (a 10-fold increase) by itself.

Materials and Methods

EMSA assay for NF-κB-DNA binding was as follows. Human Jurkat leukemiaT-cells (clone E6-1; Amer. Type Culture Collection, Rockville, Md.) weregrown in RPMI-1640 Media (Gibco-BRL, Rockville, Md.) supplemented with10% fetal bovine serum, penicillin (614 ηg/mL), streptomycin (10 μg/mL)and HEPES buffer, pH 7.2 at 37° C., 5% CO₂. The Jurkat cells (1×10⁶cells/mL) were subsequently treated with various concentrations of thecompounds for 30 minutes at 37° C. and 5% CO₂ followed by PMA (50 ng/mL)and PHA (1 mM/mL) stimulation for an additional 30 minutes. The cellswere harvested by centrifugation, washed in ice cold PBS and the nuclearextracts were prepared as previously described (Dignam, et al., Nucl.Acids Res 11: 1475-1489 (1983)). The protein concentration of theextracts was determined according to the Method of Bradford (1976) withBioRad reagents. Nuclear extracts are incubated for 20 minutes at roomtemperature with a double stranded Cy3 labeled NF-kB consensusoligonucleotide, 5′-AGTTGAGGGGACTTTC CCAGGC-3′ (SEQ ID NO:1). Thebinding mixture (25 mL) contained 10 mM HEPES-NaOH pH 7.9, 4 mMtris-HCl, pH 7.9, 6.0 mM KCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.3mg/mL bovide serum albumin and 1 mg of poly (dI.dC). The bindingmixtures (10 mg of nuclear extract protein) were incubated for 20minutes at room temperature with 0.16 μmol of Cy3 labeledoligonucleotide. The mixture was loaded on a 4% polyacrylamide gelprepared in 1× tris borate/EDTA buffer and was electrophoresed at 200 Vfor 20 minutes. After electrophoresis the gel was analyzed using aphosphorimager (Biorad FX plus) for detection of the NF-kB—DNA binding.

Inhibition of translocation with p65-ELISA assay was as follows. Thequantity of p65/p50 heterodimer that has translocated into the nucleuswas measured using a NF-κB p65 sandwich ELISA assay (Imgenex Corp.).Jurkat cells were grown to 2×10⁶ cells/mL and treated with 50 ng/mL PMAand 1 μg/mL PMA/PHA and incubated at 37° C., 5% CO₂. The cells areharvested after 30 minutes and nuclear extracts are prepared aspreviously described by Dignam and coworkers (Dignam, et al., Nucl.Acids Res 11: 1475-1489 (1983)). The NF-κB p65 sandwich ELISA kit wasthen used to monitor and quantify p65 translocation into the nucleusaccording to the manufacturers protocol. PDTC is reported to be aninhibitor of NF-κB translocation and our data confirmed that itinhibited NF-κB translocation at concentrations ranging from 100 nM to5.0 μM.

Induction of apoptosis using caspase 3/7 assay was as follows. CEM cells(CCRF-CEM); Amer. Type Culture Collection, Rockville, Md.) were grown inRPMI-1640 Media (Gibco-BRL, Rockville, Md.) supplemented with 10% fetalbovine serum, penicillin (614 ηg/mL), streptomycin (10 μg/mL) and hepesbuffer, pH 7.2 at 37° C., 5% CO₂. DMSO was used as the vector for alldrugs and added in the control experiments. Cell cultures were treatedwith 1 μM, 0.1 μM or 10 nM of the imidazolines and allowed to incubateat 37° C., 5% CO₂. An aliquote was transferred to a 96-well plate andmixed with an equal volume of Apo-ONE™ Homogenous Caspase-3/7 assay(Promega Corporation) reagent. The contents of the plate were gentlymixed and allowed to incubate for 1 hour. The fluorescence of each wellwas then measured on a Molecular Imager FX Pro at 532 nm. All reporteddata was the average of two independent experiments unless otherwiseindicated.

Example 28 Preparation of Imidazoline Benzyl Esters EDCI.HCl:1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

DMAP: Dimethyl Aminopyridine

Synthesis of1-Benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid benzyl ester [SP-2-61]

In a flame dried flask under nitrogen atmosphere1-benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid (0.1 g, 0.27 mmol) was suspended in dry methylene chloride (10 ml).The solution was cooled in an ice-bath and EDCI.HCl (0.057 g, 0.29 mmol0 was added, followed by DMAP (0.35 g, 0.29 mmol) after five minutes andstirred for 20 minutes. Benzyl alcohol (0.062 g, 0.58 mmol) was addedand mixture stirred at room temperature overnight. The reaction mixturewas washed with 2N HCl (2×20 ml), saturated sodium bicarbonate (2×20 ml)and then with brine (20 ml). The organic layer was dried over sodiumsulfate and evaporated under reduced pressure. The crude product waspurified by column silica-gel chromatography using 70% ether/hexanemixture.

SP-2-61: Yield: (0.08 g, 65%). CHCl₃; ¹H NMR (300 MHz), CDCl₃: δ 1.62(s, 3H), 3.82 (d, J=15.6 Hz, 1H), 4.34 (s, 1H), 4.43 (d, J=12.6 Hz, 1H),4.65 (d, J=12.9 Hz, 1H), 4.73 (d, J=15.6 Hz, 1H), 6.94-6.97 (m, 2H),7.06-7.24 (m, 2H), 7.26-7.37 (m, 11H), 7.49-7.51 (m, 2H), 7.74-7.75 (m,2H); ¹³C NMR (75 MHz) CDCl₃: 27.06, 49.14, 64.43, 66.66, 127.02, 127.18,127.96, 128.02, 128.09, 128.14, 128.34, 128.46, 128.53, 128.58, 128.74,128.97, 130.61, 131.03, 135.87, 136.65, 137.03, 166.62, 172.0.

Example 29

Synthesis of 1-Benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazoleacid 1-phenyl-ethyl ester SP-1-396

A well-stirred suspension of1-benzyl-4-methyl-2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylicacid (11.0 g, 0.27 mmol) in dry methylene chloride (25 ml) was made in aflame dried flask under nitrogen atmosphere and cooled in an ice-bath to0° C. To this mixture was added EDCI.HCl (0.57 g, 29 mmol), followed byDMAP (0.35 gm, 29 mmol) and stirred for 20 minutes.(R)-(+)-1-Phenyl-ethanol (0.72 g, 2 eq., 58 mmol) was added and mixturestirred overnight at room temperature. The reaction mixture was washed,2N HCl (2×20 ml), saturated sodium bicarbonate (2×20 ml), and then withbrine (20 ml). The organic layer dried over sodium sulfate andevaporated under reduced pressure. The crude product was purified bycolumn silica-gel chromatography using 70% ether/hexane mixture.

(−)-SP-1-396a (RRR): Yield: (0.34 g, 61%). [α]_(D)=−118°, c=1.2, CHCl₃;¹H NMR (500 MHz), CDCl₃: δ 1.236 (d, J=6.5, 3H), 1.62 (s, 3H), 3.83 (d,J=15.5 Hz, 1H), 4.34 (s, 1H), 4.70 (d, J=15.5 Hz, 1H), 5.32 (q, J=6.5,1H), 6.94-7.16 (m, 4H), 7.17-7.28 (m, 11H), 7.48-7.49 (t, J=3.5 Hz, 3H)7.76-7.77 (m, 2H); ¹³C NMR (125 MHz) CDCl₃: 21.8, 26.95, 48.88, 72.81,73.27, 77.61, 125.87, 127.2, 127.57, 127.7, 127.77, 128.0, 128.27,128.55, 128.64, 130.08, 131.12, 136.59, 136.64, 141.35, 165.92, 171.08;E1MS: m/z=474.1 (M+).

(+)-SP-1-396b (SSR): (0.38 g, 66%). [α]_(D)=+113°, C=1.2, CHCl₃; ¹H NMR(500 MHz), CDCl₃: δ: 0.957 (d, J=6.5, 3H), 1.61 (s, 3H), 3.77 (d, J=15.5Hz, 1H), 4.33 (s, 1H), 4.66 (d, J=15.5 Hz, 1H), 5.33 (q, J=6.5, 1H),6.92-6.94 (m, 2H), 7.17-7.29 (m, 13H), 7.47-7.48 (m, 3H) 7.73-7.75 (m,2H); ¹³C NMR (125 MHz), CDCl₃: 21.44, 26.85, 48.83, 72.74, 73.57, 77.58,126.0, 127.45, 127.54, 127.79, 127.9, 128.0, 128.14, 128.37, 128.51,128.53, 128.62, 130.03, 131.15, 136.52, 137.06, 141.77, 165.94, 170.86;EIMS: m/z=474.2 (M+).

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

We claim:
 1. A method of inhibiting a cancer, which comprises contactingsaid cancer with a multi-substituted imidazoline ester in amountssufficient to inhibit said cancer, said multi-substituted imidazolineester having the formula:

wherein R₁, R₂, and R₃, and R₄ are each aryl or arylalkyl and R₅ is analkyl group; and wherein NF-κB plays a role in the cancer.
 2. The methodof claim 1, wherein said method further comprises administering saidimidazoline ester and a chemotherapeutic drug to a mammal.
 3. The methodof claim 2, wherein the mammal is human.
 4. The method of claim 2wherein said administering is oral.
 5. The method of claim 2 whereinsaid administering is topical.
 6. The method of claim 2 wherein saidadministering is by injection.
 7. The method of claim 2 wherein saidadministering is intravenous.
 8. The method of claim 2, wherein saiddrug is a platinate.
 9. The method of claim 2, wherein said drug iscamptothecin.
 10. A method of inhibiting a cancer, which comprisesadministering to a mammal having said cancer with a multi-substitutedimidazoline ester and camptothecin in amounts sufficient to inhibit saidcancer, said multi-substituted imidazoline ester having the formula:

wherein R₁, R₂, and R₃, and R₄ are each aryl or arylalkyl and R₅ is analkyl group; and wherein NF-κB plays a role in the cancer.
 11. A methodof inhibiting a cancer, which comprises administering to a mammal havingsaid cancer with a multi-substituted imidazoline ester in amountssufficient to inhibit said cancer, said multi-substituted imidazolineester having the formula:

wherein R₁, R₂, and R₃, and R₄ are each aryl or arylalkyl and R₅ is analkyl group; and wherein NF-κB plays a role in the cancer.